Revision 1

#53898Store at -20C

1 Kit

(9 x 20 microliters)

Cell Signaling Technology

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

Support: 877-678-TECH (8324)

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For Research Use Only. Not for Use in Diagnostic Procedures.
Product Includes Product # Quantity Mol. Wt Isotype/Source
FAM134B (E8Y9R) Rabbit mAb 83414 20 µl 70 kDa Rabbit IgG
CCPG1 (E3C5G) Rabbit mAb 80158 20 µl 105-120 kDa Rabbit IgG
XBP-1s (E9V3E) Rabbit mAb 40435 20 µl 60 (human), 55 (mouse/rat) kDa Rabbit IgG
ATF-6 (D4Z8V) Rabbit mAb 65880 20 µl 90-100 kDa Rabbit IgG
Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb 3398 20 µl 38 kDa Rabbit IgG
PERK (C33E10) Rabbit mAb 3192 20 µl 140 kDa Rabbit IgG
ATF-4 (D4B8) Rabbit mAb 11815 20 µl 49 kDa Rabbit IgG
IRE1α (14C10) Rabbit mAb 3294 20 µl 130 kDa Rabbit IgG
BiP (C50B12) Rabbit mAb 3177 20 µl 78 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 ER Homeostasis Antibody Sampler Kit provides an economical means of detecting proteins involved in ER homeostasis by regulating ER stress and ER-phagy. The kit includes enough antibodies to perform 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 antibody.

Background

The endoplasmic reticulum (ER) is a large organelle extending from the nuclear envelope to the plasma membrane with diversity in structure and function (1,2). It functions in calcium storage, lipid and steroid synthesis, and protein folding, processing, and transport. ER structure and function is regulated in a dynamic fashion adapting to situations of ER stress and organelle damage (1,2). When demands on protein processing exceeds capabilities, cells trigger an adaptive mechanism called the unfolded protein response (UPR) which is largely controlled by the activities of three pathways: PERK, IRE1α, and ATF-6 (1). The ER chaperone protein BiP is recruited to unfolded proteins in the ER lumen and its dissociation from PERK, IRE1α, and ATF-6 leads to their activation. PERK is a kinase on the ER membrane that couples ER stress to changes in translation. PERK activation during ER stress leads to phosphorylation of eIF2α, repressing most translation but selectively inducing some targets such as ATF-4, a transcription factor that regulates targets in the recovery of the stress response. IRE1α is an ER protein with endoribonuclease activity that is activated during ER stress and converts XBP-1 from an unspliced XBP-1μ isoform to a spliced XBP-1s isoform functioning as a transcription factor regulating stress response genes. Lastly, during ER stress, ATF-6 is cleaved liberating a mature transcription factor controlling stress response genes.
Subsequent to ER expansion triggered by the UPR, cells may trigger a process of ER-phagy, the degradation of ER fragments through autophagy (3). Autophagy is a process for the bulk degradation of cytoplasmic components by a double membrane autophagosome fusing to the lysosome. Selective autophagy, like ER-phagy, permits the degradation of specific targets. This process generally involves specific cargo receptors containing LIR or GIM domains targeting bound cargo to the autophagosome through interactions with LC3 or GABARAP, respectively. FAM134B was the first ER-phagy receptor discovered. Loss of FAM134B can sensitize cells to apoptosis when challenged by nutrient deprivation or ER stress stimuli. CCPG1 is another ER-phagy cargo receptor that associates with FIP200, a component of the ULK1 complex facilitating ER trafficking to autophagosomes. Importantly, CCPG1 is transcriptionally regulated by ER stress. Taken together, signaling from the UPR and ER-phagy help regulate ER homeostasis. Defects in this process may contribute to pathological conditions, including metabolic and neurological disorders, cancer, and defense against infectious diseases (3).

  1. Sano, R. and Reed, J.C. (2013) Biochim Biophys Acta 1833, 3460-3470.
  2. Ferro-Novick, S. et al. (2021) Trends Biochem Sci 46, 630-639.
  3. Hübner, C.A. and Dikic, I. (2020) Cell Death Differ 27, 833-842.

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.
    U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
    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
    #53898

    ER Homeostasis Antibody Sampler Kit

    ER Homeostasis Antibody Sampler Kit: Image 1 Expand Image
    Western blot analysis of extracts from 293 and HeLa cells, untreated (-) or tunicamycin-treated (2 μg/ml, 8 hr; +), using ATF-4 (D4B8) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 2 Expand Image
    CUT&Tag was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across Ddit3/CHOP gene, a known target gene of ATF4 (see our ChIP-qPCR figure).
    ER Homeostasis Antibody Sampler Kit: Image 3 Expand Image
    CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ASNS, a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
    ER Homeostasis Antibody Sampler Kit: Image 4 Expand Image
    Western blot analysis of extracts from various cell lines using BiP (C50B12) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 5 Expand Image
    Western blot analysis of extracts from various cell types using PERK (C33E10) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 6 Expand Image
    Western blot analysis of extracts from various cell lines, using IRE1α (14C10) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 7 Expand Image
    Western blot analysis of extracts from C2C12 cells, untreated or thapsigargin-treated, using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb (upper) or eIF2α Antibody #9722 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 8 Expand Image
    Western blot analysis of extracts from 293T cells untreated (-) or treated with Tunicamycin #12819 (2 μg/ml, 8 hr; +), using XBP-1s (E9V3E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 9 Expand Image
    Western blot analysis of extracts from 293T cells, untreated (-) or treated with tunicamycin (1 mM; 1hr; +), using ATF-6 (D4Z8V) Rabbit mAb. 
    ER Homeostasis Antibody Sampler Kit: Image 10 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.
    ER Homeostasis Antibody Sampler Kit: Image 11 Expand Image
    Western blot analysis of extracts from various cell lines using CCPG1 (E3C5G) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Absence of signal in Jurkat cells is predicted by RNAseq and confirms the specificity of the antibody.
    ER Homeostasis Antibody Sampler Kit: Image 12 Expand Image
    Western blot analysis of extracts from SNU-475 cells, untreated (-) or treated with Tunicamycin #12819 (5 μg/mL, 24 hr; +), using CCPG1 (E3C5G) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 13 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human CCPG1 protein (hCCPG1-Myc/DDK; +), using CCPG1 (E3C5G) Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 14 Expand Image
    Immunoprecipitation of CCPG1 protein from Colo 205 extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CCPG1 (E3C5G) Rabbit mAb. Western blot analysis was performed using CCPG1 (E3C5G) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
    ER Homeostasis Antibody Sampler Kit: Image 15 Expand Image
    Western blot analysis of extracts from various cell lines using FAM134B (E8Y9R) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal for FAM134B in Hep G2 cells is consistent with RNAseq data and confirms the specificity of the antibody for FAM134B.
    ER Homeostasis Antibody Sampler Kit: Image 16 Expand Image
    Immunoprecipitation of ATF-4 from extracts of 293 cells, treated with tunicamycin (2 μg/ml, 8 hr), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or ATF-4 (D4B8) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using ATF-4 (D4B8) Rabbit mAb. Mouse Anti-rabbit IgG (Light-Chain Specific) (L57A3) mAb #3677 and Anti-mouse IgG, HRP-linked Antibody #7076 were used as secondary antibodies.
    ER Homeostasis Antibody Sampler Kit: Image 17 Expand Image
    CUT&Tag was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 12 (upper), including Ddit3/CHOP (lower), a known target gene of ATF-4 (see our ChIP-qPCR figure).
    ER Homeostasis Antibody Sampler Kit: Image 18 Expand Image
    CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and ATF-4 (D4B8) Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Library was prepared using DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 7 (upper), including ASNS (lower), a known target gene of ATF-4 (see additional figure containing CUT&RUN-qPCR data).
    ER Homeostasis Antibody Sampler Kit: Image 19 Expand Image
    Immunohistochemical analysis of paraffin-embedded human glioblastoma using BiP (C50B12) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 20 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phopsho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 21 Expand Image
    Western blot analysis of extracts from C2C12 cells untreated (-) or treated with Tunicamycin #12819 (2 μg/ml, 8 hr; +) and INS-1 cells untreated (-) or treated with Tunicamycin #12819 (5 μg/ml, 24 hr; +), using XBP-1s (E9V3E) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 22 Expand Image
    Immunoprecipitation of ATF-6 from 293T cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ATF-6 (D4Z8V) Rabbit mAb. Western blot analysis was performed using ATF-6 (D4Z8V) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 23 Expand Image
    Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human FAM134B protein (hFAM134B-Myc/DDK; +), using FAM134B (E8Y9R) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).
    ER Homeostasis Antibody Sampler Kit: Image 24 Expand Image
    Confocal immunofluorescent analysis of HeLa cells, untreated (left) or tunicamycin-treated (2 μg/ml, 8 hr; right), using ATF-4 (D4B8) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).
    ER Homeostasis Antibody Sampler Kit: Image 25 Expand Image
    CUT&RUN was performed with Hep G2 cells treated with Thapsigargin #12758 (300nM) for 4h and either ATF-4 (D4B8) 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 TRB3 exon 1 primer, human ASNS exon 1 primer 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.
    ER Homeostasis Antibody Sampler Kit: Image 26 Expand Image
    Immunohistochemical analysis of paraffin-embedded human colon carcinoma using BiP (C50B12) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 27 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 28 Expand Image
    Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or treated with Tunicamycin #12819 (2 µg/ml, 8 hr; right), using XBP-1s (E9V3E) Rabbit mAb (green). Actin filaments were labeled with DyLight 554 Phalloidin #13054 (red).
    ER Homeostasis Antibody Sampler Kit: Image 29 Expand Image
    Immunoprecipitation of FAM134B protein from SCLC-21H cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is FAM134B (E8Y9R) Rabbit mAb. Western blot analysis was performed using FAM134B (E8Y9R) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
    ER Homeostasis Antibody Sampler Kit: Image 30 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across Ddit3/CHOP, a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
    ER Homeostasis Antibody Sampler Kit: Image 31 Expand Image
    Immunohistochemical analysis of paraffin-embedded human hepatocellular carcinoma using BiP (C50B12) Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 32 Expand Image
    Immunohistochemical analysis of paraffin-embedded human lymphoma using Phospho-eIF2α (Ser51) (D9G8) XP® Rabbit mAb.
    ER Homeostasis Antibody Sampler Kit: Image 33 Expand Image
    Flow cytometric analysis of C2C12 cells, untreated (blue, low) or treated with tunicamycin (2 μg/mL, 8 hours; green, positive) using XBP-1s (E9V3E) 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.
    ER Homeostasis Antibody Sampler Kit: Image 34 Expand Image
    Confocal immunofluorescent analysis of TT cells (left, positive) and Hep G2 cells (right, negative) using FAM134B (E8Y9R) Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).
    ER Homeostasis Antibody Sampler Kit: Image 35 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight and ATF-4 (D4B8) Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 10 (upper), including Ddit3/CHOP (lower), a known target gene of ATF4 (see additional figure containing ChIP-qPCR data).
    ER Homeostasis Antibody Sampler Kit: Image 36 Expand Image
    Immunohistochemical analysis of paraffin-embedded human breast carcinoma using BiP (C50B12) Rabbit mAb in the presence of control peptide (left) or BiP Blocking Peptide #1084 (right).
    ER Homeostasis Antibody Sampler Kit: Image 37 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from C2C12 cells differentiated with 10% horse serum for 7 days then treated with tunicamycin (2 ug/ml) for 7 hours and either XBP-1s (E9V3E) Rabbit mAb #2392 or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human DNAJB9 Exon 1 Primers #79879, mouse EIF2AK3 promoter primers and SimpleChIP® Mouse GAPDH Intron 2 Primers #8986. 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.
    ER Homeostasis Antibody Sampler Kit: Image 38 Expand Image
    Chromatin immunoprecipitations were performed with cross-linked chromatin from mouse embryonic fibroblasts treated with tunicamycin (2ug/ml) overnight, and ATF-4 (D4B8) 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 SimpleChIP® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. 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.
    ER Homeostasis Antibody Sampler Kit: Image 39 Expand Image
    Flow cytometric analysis of A204 cells using BiP (C50B12) Rabbit mAb (blue) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.