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36422
Microglia LPS-Related Module Antibody Sampler Kit
Primary Antibodies

Microglia LPS-Related Module Antibody Sampler Kit #36422

Western Blotting Image 1

Western blot analysis of extracts from J774A.1 and Raw 264.7 cells using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 2

Western blot analysis of cell extracts from Baf3, 32D, and mouse spleen using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

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Western Blotting Image 3

Western blot analysis of extracts from HeLa, Hep G2, and BaF3 cells using Rab11FIP1 (D9D8P) Rabbit mAb.

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Western Blotting Image 4

Western blot analysis of extracts from various cell lines using Integrin α4 (D2E1) XP® Rabbit mAb.

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Western Blotting Image 5

Western blot analysis of extracts from various cell lines using IQGAP1 (D8K4X) XP® Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from HeLa, NIH/3T3, and C6 cells, untreated or staurosporine-treated (1 µM), and Jurkat cells, untreated or etoposide-treated (25 µM), using Cleaved Lamin A (Small Subunit) (30H5) Mouse mAb.

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Western Blotting Image 7

Western blot analysis of extracts from Raw 264.7 and KNRK cell lines using IKKε (D61F9) XP® Rabbit mAb.

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Lamin A/C (4C11) Mouse mAb.

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Western Blotting Image 9

Western blot analysis of various cell extracts, using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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Western Blotting Image 10

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.

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IP Image 11

Immunoprecipitation of ASC/TMS1 from J774A.1 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is ASC (D2W8U) Rabbit mAb (Mouse Specific). Western blot analysis was performed using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific).

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IHC-P (paraffin) Image 12

Immunohistochemical analysis of paraffin-embedded mouse spleen using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

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Western Blotting Image 13

Western blot analysis of extracts from MCF7 and SNB19 cells using Rab11FIP1 (D9D8P) Rabbit mAb and β-Actin (D6A8) Rabbit mAb #8457.

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Flow Cytometry Image 14

Flow cytometric analysis of Jurkat cells using Integrin α4 (D2E1) XP® 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.

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IHC-P (paraffin) Image 15

breast:

Immunohistochemical analysis of paraffin-embedded human infiltrating papillary carcinoma of the breast using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IF-IC Image 16

Immunofluorescent analysis of HeLa cells, untreated (right) or staurosporine-treated (left), using Cleaved Lamin A (Small Subunit) (30H5) Mouse mAb.

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Flow Cytometry Image 17

Flow cytometric analysis of Raw 264.7 cells using IKKε (D61F9) XP® 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.

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Western Blotting Image 18

Western blot analysis of extracts from THP-1 cells, untreated or treated with cycloheximide (CHX, 10 μg/ml, overnight) followed by TNF-α #8902 (20 ng/ml, 4 hr), using Lamin A/C (4C11) Mouse mAb.

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Western Blotting Image 19

Western blot analysis of Staurosporine #9953 treated (1 μM, 3 hr) or untreated HeLa cells using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (upper) and Ezrin/Radixin/Moesin Antibody #3142 (lower).

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Flow Cytometry Image 20

Flow cytometric analysis of Raw264.7 cells (blue) and J774A.1 cells (green) using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (solid lines) or a 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.

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded LL2 syngeneic tumor using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

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IP Image 22

Immunoprecipitation of Rab11FIP1 from HeLa cell extracts using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Rab11FIP1 (D9D8P) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Rab11FIP1 (D9D8P) Rabbit mAb.

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IF-IC Image 23

Confocal immunofluorescent analysis of Jurkat (positive; left) and K-562 (negative; right) cells using Integrin α4 (D2E1) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded SK-MEL-28 (left) and LNCaP (right) cell pellets using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IF-IC Image 25

Confocal immunofluorescent analysis of RAW 264.7 (left) and C2C12 (right) cells using IKKepsilon (D61F9) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® (fluorescent DNA dye).

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IHC-P (paraffin) Image 26

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Lamin A/C (4C11) Mouse mAb.

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded human breast ductal carcinoma using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IF-IC Image 28

Confocal immunofluorescent analysis of mouse primary bone marrow-derived macrophages (BMDMs) either untreated (upper left) or treated with LPS (50 ng/ml, 4 hr, middle) or LPS followed by ATP (5 mM, 45 min, upper right), and J774A.1 (lower left) or Raw 264.7 (lower right) cells, using ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Note the translocation of ASC to inflammasomes following stimulation with LPS and ATP (white arrows).

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Flow Cytometry Image 29

Flow cytometric analysis of NIH/3T3 cells (red) and 32D cells (blue), using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific).

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IF-IC Image 30

Confocal immunofluorescent analysis of MCF7 (positive, left) and SNB19 (negative, right) cells using RAB11FIP1 (D9D8P) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 31

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using IQGAP1 (D8K4X) XP® Rabbit mAb.

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IHC-P (paraffin) Image 32

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Lamin A/C (4C11) Mouse mAb.

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IHC-P (paraffin) Image 33

Immunohistochemical analysis of paraffin-embedded human ovarian endometrioid adenocarcinoma using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IF-IC Image 34

Confocal immunofluorescent analysis of 32D cells (left) and C2C12 cells (right), using HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 35

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using IQGAP1 (D8K4X) XP® Rabbit mAb.

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Flow Cytometry Image 36

Flow cytometric analysis of HeLa cells (green) using Lamin A/C (4C11) Mouse mAb (solid lines) or a concentration matched Mouse (G3A1) mAb IgG Isotype Control #5415 (dashed lines). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.

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IHC-P (paraffin) Image 37

Immunohistochemical analysis of paraffin-embedded human gastric adenocarcinoma, untreated (left) or λ phosphatase treated (right), using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb.

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IF-F Image 38

Confocal immunofluorescent analysis of mouse Tg2576 brain which overexpresses mutant human APP695. Sections were first labeled with HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) (green) and APP/β-Amyloid (NAB228) Mouse mAb #2450 (yellow). After blocking free secondary binding sites with Mouse (G3A1) mAb IgG1 Isotype Control #5415, sections were incubated with GFAP (GA5) Mouse mAb (Alexa Fluor® 647 Conjugate) #3657 (red). Nuclei were labeled with Hoechst 33342 #4082 (blue).

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IF-IC Image 39

Confocal immunofluorescent analysis of A549 (left) and Hep G2 (right) cells using IQGAP1 (D8K4X) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 40

Confocal immunofluorescent analysis of HeLa cells using Lamin A/C (4C11) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red).

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IF-IC Image 41

Confocal immunofluorescent analysis of HeLa cells, untreated (left), treated with Staurosporine #9953 (1 μM, 1hr; center), or λ phosphatase (right) labeled with Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 phalloidin (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

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IF-F Image 42

Immunofluorescent analysis of normal rat brain using Lamin A/C (4C11) Mouse mAb (green) and MAP2 Antibody #4542 (red).

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ELISA-Peptide Image 43

Validation of Phospho-Ezrin (Thr567)/ Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb (ELISA Specific) in peptide DELFIA® assay using phospho- and nonphospho-peptide controls, and DELFIA® secondary antibodies (available from Perkin Elmer Life and Analytical Sciences).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) 67824 20 µl
  • WB
  • IP
  • IF
  • F
M 22 Rabbit IgG
HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) 3892 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
M R 80 Rabbit IgG
Rab11FIP1 (D9D8P) Rabbit mAb 12849 20 µl
  • WB
  • IP
  • IF
H M Mk 85 Rabbit IgG
Integrin α4 (D2E1) XP® Rabbit mAb 8440 20 µl
  • WB
  • IP
  • IF
  • F
H M R 70, 140, 150, Rabbit IgG
IQGAP1 (D8K4X) XP® Rabbit mAb 20648 20 µl
  • WB
  • IHC
  • IF
H M R Mk 195 Rabbit IgG
Cleaved Lamin A (Small Subunit) (30H5) Mouse mAb 2036 20 µl
  • WB
  • IF
H M R 28 Mouse IgG1
IKKε (D61F9) XP® Rabbit mAb 3416 20 µl
  • WB
  • IP
  • IF
  • F
M R 80 Rabbit 
Lamin A/C (4C11) Mouse mAb 4777 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 74 (Lamin A), 63 (Lamin C) Mouse IgG2a
Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb 3726 20 µl
  • WB
  • IHC
  • IF
H M R Mk 75 Moesin. 80 Ezrin, Radixin. Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Microglia LPS-Related Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of LPS-related microglial activity by western blot and/or immunofluorescence.

Each antibody in the Microglia LPS-Related Module Antibody Sampler Kit detects endogenous levels of its target protein. HS1 (D5A9) XP® Rabbit mAb (Rodent Specific) does not recognize human HS1 protein. HS1 has a calculated size of 54 kDa, but has an apparent molecular weight of 80 kDa on SDS-PAGE gels. Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) (48G2) Rabbit mAb recognizes endogenous levels of Ezrin, Radixin, and Moesin only when phosphorylated at Thr567, Thr564, and Thr558 respectively. Lamin A/C (4C11) Mouse mAb detects endogenous levels of lamin A and lamin C proteins and also reacts with the larger fragments of lamin A (50 kDa) and lamin C (41 kDa) produced by caspase cleavage during apoptosis. Cleaved Lamin A (Small Subunit) (30H5) Mouse mAb detects endogenous levels of the small fragment of lamin A (and lamin C) resulting from cleavage at Asp230 and does not cross-react with full length lamin A or C.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu310 of mouse HS1, Leu248 of human Rab11FIP1, Ser1027 of human integrin α4, Thr567 of human ezrin, Asp230 of human lamin A, the amino terminus of human IQGAP1, the carboxy terminus of mouse IKKε, and a recombinant fragment of human lamin A and mouse ASC/TMS1 protein.

Distinct microglial activation states have been identified using RNA-seq data from a vast array of neurological disease and aging models. These activation states have been categorized into modules corresponding to proliferation, neurodegeneration, interferon-relation, LPS-relation, and many others (1). Previous work identifying markers of specific brain cell types using RNA-seq has shown HS1 and ASC/TMS1 to be useful and specific tools to study microglia (2). HS1 is a protein kinase substrate that is expressed only in tissues and cells of hematopoietic origin (3) and ASC/TMS1 has been found to be a critical component of inflammatory signaling where it associates with and activates caspase-1 in response to pro-inflammatory signals (4).

The Rab11-family interacting proteins (Rab11-FIPs) facilitate Rab11-dependent vesicle recycling through interaction with the conserved carboxyl terminal Rab11 binding domain (5,6). Rab11FIP1 has been shown to play a role in endocytic sorting and trafficking of EGFR and integrin subunits (6). Integrins are α/β heterodimeric cell surface receptors that mediate cell adhesion and migration and regulate cell growth and survival. Two significant α4 integrins, α4β1 and α4β7, interact with VCAM-1, fibronectin, and MAdCAM-1 at cell adhesions and have been shown to play an important role in cell trafficking during inflammatory processes (7-9). Lamins are nuclear membrane structural components important for maintaining normal cell functions. Lamin A/C is cleaved by caspase-6 and serves as a marker for caspase-6 activation. The cleavage of lamins results in nuclear dysregulation and cell death (10,11). The ezrin, radixin, and moesin (ERM) proteins function as linkers between the plasma membrane and the actin cytoskeleton and are involved in cell adhesion, membrane ruffling, and microvilli formation (12). ERM proteins undergo intra or intermolecular interaction between their amino- and carboxy-terminal domains, existing as inactive cytosolic monomers or dimers (13). Phosphorylation at a carboxy-terminal threonine residue (Thr567 of ezrin, Thr564 of radixin, Thr558 of moesin) disrupts the amino- and carboxy-terminal association and may play a key role in regulating ERM protein conformation and function (14,15). IQGAPs are scaffolding proteins involved in mediating cytoskeletal function that contain multiple protein interaction domains (16). IQGAP1 is ubiquitously expressed and has been found to interact with APC (17) and the CLIP170 complex in response to small GTPases, promoting cell polarization and migration (18).  IKKε is an IKK-related kinase that functions as part of the signal-stimulated noncanonical pathway of NF-kB activation (19). IKKε plays a role in the immune response and also impacts cell proliferation and transformation (20).

  1. Friedman, B.A. et al. (2018) Cell Rep 22, 832-47.
  2. Zhang, Y. et al. (2014) J Neurosci 34, 11929-47.
  3. Kitamura, D. et al. (1995) Biochem Biophys Res Commun 208, 1137-46.
  4. Srinivasula, S.M. et al. (2002) J Biol Chem 277, 21119-22.
  5. Baetz, N.W. and Goldenring, J.R. (2013) Mol Biol Cell 24, 643-58.
  6. Kummer, C. and Ginsberg, M.H. (2006) Biochem Pharmacol 72, 1460-8.
  7. Sun, S.C. et al. (2013) Trends Immunol 34, 282-9.
  8. Verhelst, K. et al. (2013) Biochem Pharmacol 85, 873-80.
  9. Liu, S. et al. (2000) J Cell Sci 113 (Pt 20), 3563-71.
  10. Tsukita, S. and Yonemura, S. (1999) J Biol Chem 274, 34507-10.
  11. Briggs, M.W. and Sacks, D.B. (2003) EMBO Rep 4, 571-4.
  12. Hood, J.D. and Cheresh, D.A. (2002) Nat Rev Cancer 2, 91-100.
  13. Mangeat, P. et al. (1999) Trends Cell Biol 9, 187-92.
  14. Matsui, T. et al. (1998) J Cell Biol 140, 647-57.
  15. Gautreau, A. et al. (2000) J Cell Biol 150, 193-203.
  16. Watanabe, T. et al. (2004) Dev Cell 7, 871-83.
  17. Fukata, M. et al. (2002) Cell 109, 873-85.
  18. Hales, C.M. et al. (2001) J Biol Chem 276, 39067-75.
  19. Oberhammer, F.A. et al. (1994) J Cell Biol 126, 827-37.
  20. Rao, L. et al. (1996) J Cell Biol 135, 1441-55.
Entrez-Gene Id
66824 , 7430 , 3059 , 9641 , 3676 , 8826 , 4000 , 4478 , 80223 , 5962
Swiss-Prot Acc.
Q9EPB4 , P15311 , P14317 , Q14164 , P13612 , P46940 , P02545 , P26038 , Q6WKZ4 , P35241
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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