Revision 1

#92516Store at -20C

1 Kit

(9 x 20 microliters)

Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
LRP1 (E2Q7S) Rabbit mAb 26387 20 µl 85 kDa Rabbit IgG
ApoE (pan) (D7I9N) Rabbit mAb 13366 20 µl 35 kDa Rabbit IgG
PSD95 (D27E11) XP® Rabbit mAb 3450 20 µl 95 kDa Rabbit IgG
Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb 45737 20 µl 95 kDa Rabbit IgG
Synapsin-1 (D12G5) XP® Rabbit mAb 5297 20 µl 77 kDa Rabbit IgG
AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb 13185 20 µl 100 kDa Rabbit IgG
Phospho-AMPA Receptor 1 (GluA1) (Ser831) (A5O2P) Rabbit mAb 75574 20 µl 100 kDa Rabbit IgG
CREB (48H2) Rabbit mAb 9197 20 µl 43 kDa Rabbit IgG
Phospho-CREB (Ser133) (87G3) Rabbit mAb 9198 20 µl 43 kDa Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl Goat 

Please visit cellsignal.com for individual component applications, species cross-reactivity, dilutions, protocols, and additional product information.

Description

The ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit provides an economical means of detecting key synaptic signaling pathways in response to ApoE-mediated LRP1 activation by western blot. The kit includes enough antibodies to perform at least two western blot experiments with each primary antibody.

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibodies.

Background

Low density lipoprotein receptor related protein 1 (LRP1) is a type I transmembrane receptor that mediates the endocytosis of various ligands, including apolipoproteins and tau. Both proteins are genetically and pathologically linked to Alzheimer’s disease (AD) (1,2). Human apolipoprotein E (ApoE) is a component of circulating lipoproteins when three human genetic ApoE variants, ApoE2, ApoE3, and ApoE4, exhibit distinct receptor-binding properties and differentially contribute to AD progression through a cellular mechanism that is poorly understood (2). Altered synaptic signaling is one proposed mechanism that contributes to altered neuronal function, which correlates with disease (3-5). Postsynaptic Density protein 95 (PSD95) is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins that functions as a scaffolding protein to promote assembly and function of the postsynaptic density complex (6,7). At the presynapse, synapsins function to regulate neurotransmitter release (8,9). Dynamic phosphorylation of PSD95 at Ser295 reflects synaptic signaling that may alter synaptic function (10). In addition to PSD95, postsynaptic glutamate receptors, including AMPA-(α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors, can be directly phosphorylated. Phosphorylation of AMPA Receptor 1 (GluA1) at either Ser831 or Ser845 alters AMPA receptor ion channel function to change synaptic efficacy (11). CREB is a bZIP transcription factor that activates target genes through cAMP response elements. CREB is activated by phosphorylation at Ser133 by various signaling pathways including Erk, Ca2+,stress signaling, as well as synaptic signaling (3). 

  1. Rauch, J.N. et al. (2020) Nature 580, 381-385.
  2. Corder, E.H. et al. (1993) Science 261, 921-3.
  3. Yong, S.M. et al. (2014) Sci Rep 4, 6580.
  4. Huang, Y.A. et al. (2019) J Neurosci 39, 7408-7427.
  5. Lane-Donovan, C. and Herz, J. (2017) Trends Endocrinol Metab 28, 273-284.
  6. Cao, J. et al. (2005) J Cell Biol 168, 117-26.
  7. Chetkovich, D.M. et al. (2002) J Neurosci 22, 6415-25.
  8. Greengard, P. Mol Neurobiol 1, 81-119.
  9. Hosaka, M. et al. (1999) Neuron 24, 377-87.
  10. Kim, M.J. et al. (2007) Neuron 56, 488-502.
  11. Lee, H.K. et al. (2000) Nature 405, 955-9.

Background References

    Trademarks and Patents

    Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
    XP is a registered trademark of Cell Signaling Technology, Inc.
    All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

    Limited Uses

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    Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

    Revision 1
    #92516

    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit

    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 1 Expand Image
    Confocal immunofluorescent analysis of fixed frozen hippocampus from C57BL/6 (left) and APOE4 knock-in (hu/hu, model #1549-F, right) mice labeled with ApoE (pan) (D7I9N) Rabbit mAb (top, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to colabeling with GFAP (GA5) Mouse mAb (Alexa Fluor® 594 Conjugate) #8152 (bottom, red), and AQP4 (D1F8E) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #89851 (bottom, blue pseudocolor). Mice from Taconic Biosciences, Inc.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 2 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from mouse brain using PSD95 (D27E11) XP® Rabbit mAb #3450. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 3 Expand Image
    Simple WesternTM analysis of lysates (0.75 mg/mL) from Mouse Brain cells using Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb #45737. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the JessTM Simple Western instrument from ProteinSimple, a BioTechne brand, using the 66-440 kDa separation module.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 4 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from CAD cells using Synapsin-1 (D12G5) XP Rabbit mAb #5297. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 5 Expand Image
    CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and either CREB (48H2) Rabbit mAb or Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across NR4A3 gene.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 6 Expand Image
    Simple Western™ analysis of lysates (0.1 mg/mL) from SK-N-MC cells treated with Forskolin (30uM, 30min) and IBMX (0.5mM, 30 min) using Phospho-CREB (Ser133) (87G3) Rabbit mAb #9198. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. This antibody also detects the phosphorylated form of the CREB-related protein, ATF-1 (as indicated). The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from mouse brain, rat brain, and rat prefrontal cortex tissues using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of cell extracts from Hep G2 cells treated with Brefeldin A #9972 (10 ng/ml, 90 min) and human cerebellum using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 9 Expand Image
    Immunohistochemical analysis of paraffin-embedded human kidney using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 10 Expand Image
    Western blot analysis of extracts from various cell lines and tissues using LRP1 (E2Q7S) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from human cerebellum and rat brain using PSD95 (D27E11) XP® Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from mouse brain, untreated (-) or λ phosphatase-treated (+), using Phospho-PSD95 (Ser295) (A8F8Z) Rabbit mAb (upper) or PSD95 (D74D3) XP® Rabbit mAb #3409 (lower).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from CAD cells and neonatal mouse brain using Synapsin-1 (D12G5) XP® Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 14 Expand Image
    After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 15 Expand Image
    Western Blot analysis of extracts from SK-N-MC, COS, NIH/3T3, C6 and Drosophila S2 cells, using CREB (48H2) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 16 Expand Image
    Western blot analysis of extracts from SK-N-MC cells, untreated or forskolin- and FGF-treated, using Phospho-CREB (Ser133) (87G3) Rabbit mAb (upper) or CREB (48H2) Rabbit mAb #9197 (lower).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 17 Expand Image
    Western blot analysis of cell extracts from 293T cells, treated with Brefeldin A #9972 (10 ng/ml, 90 min; +) and mock transfected (-) or transfected with a construct expressing full-length human ApoE2 (hApoE2; +), ApoE3 (hApoE3; +), or ApoE4 (hApoE4; +), using ApoE (pan) (D7I9N) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 18 Expand Image
    Immunohistochemical analysis of paraffin-embedded liver (left) or brain (right) from C57BL/6NTac (wt/wt, model #B6-F, top) and APOE4 knock-in (hu/hu, model #1549-F, bottom) mice using ApoE (pan) (D7I9N) Rabbit mAb. Mice from Taconic Biosciences, Inc.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 19 Expand Image
    Confocal immunofluorescent analysis of MEF-1 cells (left, LRP1 (+/+), positive) or PEA 13 cells (right, LRP1 (-/-), negative) using LRP1 (E2Q7S) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 20 Expand Image
    Confocal immunofluorescent analysis of rat cerebellum and retina using PSD95 (D27E11) XP® Rabbit mAb (red) and Neurofilament-L (DA2) Mouse mAb #2835 (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 21 Expand Image
    Immunohistochemical analysis of paraffin-embedded human cerebellum using Synapsin-1 (D12G5) XP® Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 22 Expand Image
    Western blot analysis of extracts from mouse brain, untreated (-) or λ-phosphatase-treated (+), using Phospho-AMPA Receptor 1 (GluA1) (Ser831) (A5O2P) Rabbit mAb (upper) and AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb #13185 (lower).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 23 Expand Image
    Immunoprecipitation of CREB from SK-N-MC extracts. Lane 1 is CREB (48H2) Rabbit mAb, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is 10% input. Western blot analysis was perfomed using CREB (86B10) Mouse mAb #9104. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 24 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, showing nuclear staining, using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 25 Expand Image
    Immunoprecipitation of AMPA Receptor 1 (GluA1) from mouse brain extracts, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 26 Expand Image
    Immunoprecipitation of ApoE from Hep G2 cells treated with Brefeldin A #9972 (10 ng/ml, 90 min), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ApoE (pan) (D7I9N) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human spleen using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 28 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse cortex using Synapsin-1 (D12G5) XP® Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 29 Expand Image
    Immunohistochemical analysis of paraffin-embedded human astrocytoma, using CREB (48H2) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 30 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse lung using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded human small intestine using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded rat retina using Synapsin-1 (D12G5) XP® Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 33 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using CREB (48H2) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 34 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-CREB (Ser133) (87G3) Rabbit mAb in the presence of control peptide (left) or Phospho-CREB (Ser133) Blocking Peptide #1090 (right).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 35 Expand Image
    Confocal immunofluorescent analysis of mouse hippocampus using AMPA Receptor 1 (GluA1) (D4N9V) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human skin using ApoE (pan) (D7I9N) Rabbit mAb in the presence of control peptide (left) and antigen-specific peptide (right).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 37 Expand Image
    Confocal immunofluorescent analysis of mouse brain using Synapsin-1 (D12G5) XP® Rabbit mAb (green) and β3-Tubulin (TU-20) Mouse mAb #4466 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 38 Expand Image
    Immunohistochemical analysis of paraffin-embedded human Non-Hodgkin's lymphoma, using CREB (48H2) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 39 Expand Image
    Immunohistochemical analysis of paraffin-embedded SK-N-MC cells, untreated (left) or IBMX- and forskolin-treated (right), showing induced nuclear staining, using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 40 Expand Image
    Immunohistochemical analysis of paraffin-embedded 293T cell pellets, control (left-top) or transfected with human ApoE isoforms ApoE2 (right-top), ApoE3 (left-bottom), and ApoE4 (right-bottom) using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 41 Expand Image
    Immunohistochemical analysis of paraffin-embedded mouse brain, using CREB (48H2) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 42 Expand Image
    Immunohistochemical analysis of paraffin-embedded human renal cell carcinoma, untreated (left) or lambda phosphatase-treated (right), using Phospho-CREB (Ser133) (87G3) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 43 Expand Image
    Immunohistochemical analysis of paraffin-embedded Hep G2 cell pellet (left, positive) or 293T cell pellet (right, negative) using ApoE (pan) (D7I9N) Rabbit mAb.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 44 Expand Image
    Confocal immunofluorescent analysis of mouse cerebellum labeled with CREB (48H2) Rabbit mAb (red) and Neurofilament-L (DA2) Mouse mAb #2835 (green). Blue pseudocolor =DRAQ5® #4084 (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 45 Expand Image
    Confocal immunofluorescent images of rat dentate gyrus, either sham-operated (left) or 15 min ischemia followed by 30 min (center) and 4 h (right) reperfusion, labeled with Phospho-CREB (Ser133) (87G3) Rabbit mAb (red), Neurofilament-L (DA2) Mouse mAb #2835 (blue) and Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb (Alexa Fluor® 488 Conjugate) #4854.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 46 Expand Image
    Confocal immunofluorescent analysis of Hep G2 cells (left, positive) and 293T cells (right, negative) using ApoE (pan) (D7I9N) Rabbit mAb #13366 (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 47 Expand Image
    Confocal immunofluorescent analysis of SK-N-MC cells showing nuclear stain with CREB (48H2) Rabbit mAb (A, red) compared to an isotype control (B). Blue pseudocolor =DRAQ5® (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 48 Expand Image
    Confocal microscopic images of SK-N-MC cells showing nuclear stain after 25 minute treatment with Forskolin and IBMX using Phospho-CREB (Ser133) (87G3) Rabbit mAb (left, red) compared to untreated cells (right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 49 Expand Image
    Flow cytometric analysis of Jurkat cells using CREB (48H2) Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 50 Expand Image
    Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with TPA #4174 (500nM) and Ionomycin #9995 (1uM,4hrs;green) using Phospho-CREB (Ser133) (87G3) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 51 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or CREB (48H2) Rabbit mAb (#9197), using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NR4A3, a known target gene of both Phospho-CREB and CREB (see additional figure containing ChIP-qPCR data).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 52 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells, treated with Forskolin #3828 (30 μM) for 1h and either 10 μl of CREB (48H2) Rabbit mAb or 2 μl of Normal Rabbit IgG #2729, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 53 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or CREB (48H2) Rabbit mAb (#9197), using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 9 (upper), including NR4A3 (lower), a known target gene of both Phospho-CREB and CREB (see additional figure containing ChIP-qPCR data).
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 54 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers, SimpleChIP® Human NR4A3 Promoter Primers #4829, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 55 Expand Image
    Western blot analysis of extracts from HepG2 cells (lane 1), 293T mock transfected (lane 2) or transiently transfected with a construct expressing ApoE4 (lane 3), whole liver extracts from wild type C57BL/6NTac model #B6-F mice (lane 4), or ApoE4 knock-in (model #1549-F) (lane 5), whole brain extracts from wild type C57BL/6NTac mice (lane 6), or ApoE4 knock-in (lane 7) using ApoE (pan) (D7I9N) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Mice from Taconic Biosciences, Inc.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 56 Expand Image
    CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and Phospho-CREB (Ser133) (87G3) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across NR4A3 gene.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 57 Expand Image
    CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and Phospho-CREB (Ser133) (87G3) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-Seq, CUT&RUN) #56795. The figures show binding across chromosome 9 (upper), including NR4A3 (lower) gene.
    ApoE Synaptic Formation and Signaling Pathway Antibody Sampler Kit: Image 58 Expand Image
    CUT&RUN was performed with 293 cells treated with Forskolin #3828 (30 μM) for 1h and either Phospho-CREB (Ser133) (87G3) Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human ALS2 exon 1 primers and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.