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93195
Microglia Neurodegeneration Module Antibody Sampler Kit
Primary Antibodies

Microglia Neurodegeneration Module Antibody Sampler Kit #93195

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 various cell lines using Cathepsin B (D1C7Y) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Chromatin IP-seq Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across ARRDC3, a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

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Western Blotting Image 5

Western blot analysis of extracts from Hep G2 cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +), Raji cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +) and U-2 OS cells untreated (-) or treated with DMOG (1 mM, 6 h; +) using HIF-1α (D1S7W) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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IF-IC Image 6

Confocal immunofluorescent analysis of HeLa cells, treated with either 10 μM MG132 (left) or 10 μM MG132 and 1 mM DMOG (right), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red).

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Western Blotting Image 7

Western blot analysis of extracts from HeLa cells, treated with either 10 μM of MG132 (to accumulate hydroxylated HIF-1α) or 10 µM MG132 and 1 mM DMOG (to accumulate nonhyroxylated HIF-1α), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (upper) or total HIF-1α Antibody #3716 (lower).

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Western Blotting Image 8

Western blot analysis of extracts from various cell lines using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines using Axl (C89E7) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

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IHC-P (paraffin) Image 10

Immunohistochemical analysis of paraffin-embedded human tonsil using CD68 (D4B9C) XP® Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of extracts from THP-1, U-937, and Jurkat cells using CD68 MultiMab™ Rabbit mAb mix (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 12

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 13

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 14

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

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Western Blotting Image 15

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing full-length human Cathepsin B (hCTSB; +) or mouse Cathespin B (mCTSB; +) using Cathepsin B (D1C7Y) XP® Rabbit mAb.

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Chromatin IP Image 16

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM, overnight) and either HIF-1α (D1S7W) XP® Rabbit mAb 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 human ARRDC3 downstream primers, SimpleChIP® Human ERRFI1 Upstream Primers #31180, 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.

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IP Image 17

Immunoprecipitation of HIF-1α from lysate of Hep G2 cells treated with cobalt chloride (100 µM, 4 h). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HIF-1α (D1S7W) XP® Rabbit mAb. Western blot analysis was performed using HIF-1α (D1S7W) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

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IP Image 18

Immunoprecipitation of Galectin-3/LGALS3 from SK-MEL-28 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb (lane 3).

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded breast carcinoma using Axl (C89E7) Rabbit mAb. Note staining of infiltrating cells.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human serous papillary carcinoma of the ovary using CD68 (D4B9C) XP® Rabbit mAb.

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IP Image 21

Immunoprecipitation of CD68 from U-937 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is CD68 MultiMab™ Rabbit mAb mix. Western blot analysis was performed using CD68 MultiMab™ Rabbit mAb mix.

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Flow Cytometry Image 22

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 23

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

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

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Flow Cytometry Image 25

Flow cytometric analysis of U-2 OS cells, untreated (blue) or treated with DMOG (1 mM, 6 h; green), using HIF-1α (D1S7W) XP® 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.

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IHC-P (paraffin) Image 26

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

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IHC-P (paraffin) Image 27

Immunohistochemical analysis of paraffin-embedded metastatic lung carcinoma using Axl (C89E7) Rabbit mAb.

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IHC-P (paraffin) Image 28

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using CD68 (D4B9C) XP® Rabbit mAb.

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IF-IC Image 29

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 30

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

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IHC-P (paraffin) Image 31

Immunohistochemical analysis of paraffin-embedded normal human kidney using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

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IF-IC Image 32

Confocal immunofluorescent analysis of Hep G2 cells, untreated (left) or treated with cobalt chloride (500 μM, 24 h; right), using HIF-1α (D1S7W) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

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IHC-P (paraffin) Image 33

Immunohistochemical analysis of paraffin-embedded SK-MEL-28 (left) and LNCaP (right) cell pellets using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb.

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IHC-P (paraffin) Image 34

Immunohistochemical analysis of paraffin-embedded cell pellets, NCI-H1299 (left) or Jurkat (right), using Axl (C89E7) Rabbit mAb.

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IHC-P (paraffin) Image 35

Immunohistochemical analysis of paraffin-embedded THP-1 (left) and Jurkat (right) cell pellets using CD68 (D4B9C) XP® Rabbit mAb.

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IF-F Image 36

Confocal immunofluorescent analysis of mouse Tg2576 brain which overexpresses mutant human APP695. Sections were first labeled with ASC/TMS1 (D2W8U) Rabbit mAb (Mouse Specific) #67824 (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 37

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 38

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Cathepsin B (D1C7Y) XP(R) Rabbit mAb.

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IHC-P (paraffin) Image 39

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Flow Cytometry Image 40

Flow cytometric analysis of Jurkat cells (blue) and DU145 cells (green) using Axl (C89E7H4) Rabbit mAb.

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IHC-P (paraffin) Image 41

Multiplex immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using CD68 (D4B9C) XP® rabbit mAb (red), PD-1 (EH33) mouse mAb #43248 (green), CD8α (C8/144B) mouse mAb #70306 (magenta), Pan-keratin (C11) mouse mAb #4545 (cyan), LAG3 (D2G4O™) XP® rabbit mAb #15372 (orange), and TIM-3 (D5D5R™) XP® rabbit mAb #45208 (yellow).

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IF-F Image 42

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 43

Confocal immunofluorescent analysis of SK-MEL-28 (left) and LNCaP (right) cells, using Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC Image 44

Confocal immunofluorescent analysis of DU 145 (left) and HCC827 (right) cells using Axl (C89E7) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 45

Multiplex immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using CD68 (D4B9C) XP® rabbit mAb (orange), PD-L1 (E1L3N®) XP® rabbit mAb #13684 (red), PD-L2 (D7U8C™) XP® rabbit mAb (magenta) #82723, Arginase-1 (D4E3M™) XP® rabbit mAb #93668 (green), IDO (D5J4E™) rabbit mAb #86630 (yellow), and Pan-keratin (C11) mouse mAb #4545 (cyan).

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Flow Cytometry Image 46

Flow cytometric analysis of Daudi cells (blue) and U266 cells (green) using Cathepsin B (D1C7Y) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 47

Flow cytometric analysis of Jurkat cells (blue) and THP-1 cells (green) using CD68 (D4B9C) XP® Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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IF-IC Image 48

Confocal immunofluorescent analysis of Malme-3M (left) and MCF7 (right) cells using Cathepsin (D1C7Y) XP® Rabbit mAb (green) and β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Flow Cytometry Image 49

Flow cytometric analysis of live human peripheral blood mononuclear cells co-stained with anti-human CD14 using CD68 (D4B9C) XP® Rabbit mAb (right) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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Flow Cytometry Image 50

Flow cytometric analysis of fixed and permeabilized human peripheral blood mononuclear cells co-stained with anti-human CD14 using CD68 (D4B9C) XP® Rabbit mAb (right) compared to a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (left). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

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IF-IC Image 51

Confocal immunofluorescent analysis of THP-1 (left) and Jurkat (right) cells using CD68 (D4B9C) XP® Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Projected images from a z-stack series are shown.

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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
Cathepsin B (D1C7Y) XP® Rabbit mAb 31718 20 µl
  • WB
  • IHC
  • IF
  • F
H M R 44, 27, 24 Rabbit IgG
HIF-1α (D1S7W) XP® Rabbit mAb 36169 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M Mk 120 Rabbit IgG
Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb 3434 20 µl
  • WB
  • IP
  • IF
H Mk 120 Rabbit IgG
Galectin-3/LGALS3 (D4I2R) XP® Rabbit mAb 87985 20 µl
  • WB
  • IP
  • IHC
  • IF
H 28 Rabbit IgG
Axl (C89E7) Rabbit mAb 8661 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 138 Rabbit IgG
CD68 (D4B9C) XP® Rabbit mAb 76437 20 µl
  • IHC
  • IF
  • F
H Rabbit IgG
CD68 MultiMab™ Rabbit mAb mix 86985 20 µl
  • WB
  • IP
H 70-80, 130-140 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Microglia Neurodegeneration Module Antibody Sampler Kit provides an economical means of detecting proteins identified as markers of microglial activity during neurodegeneration by western blot and/or immunofluorescence.

Each antibody in the Microglia Neurodegeneration 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. Cathepsin B (D1C7Y) XP® Rabbit mAb recognizes endogenous levels of total cathepsin B protein and detects the heavy chain subunit of cathepsin B. Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb detects endogenous levels of HIF-1α only when hydroxylated at Pro564 and may cross-react with other overexpressed proline hydroxylated proteins. Axl (C89E7) Rabbit mAb does not cross-react with Tyro3.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu310 of mouse HS1, Leu478 of human HIF-1α, the amino terminus of human Galectin-3/LGALS3, a hydroxypeptide surrounding Pro564 of human HIF-1α, and recombinant proteins specific to mouse ASC/TMS1, human Axl, human CD68, and the heavy chain subunit of human cathepsin B protein.

MultiMab™ rabbit monoclonal mix antibodies are prepared by combining individual rabbit monoclonal clones in optimized ratios for the approved applications. This product is optimized to detect CD68 as a monomer and a dimer by western blot and was produced by immunizing animals with recombinant human CD68 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).

CD68 is a common marker for macrophage lineage cells; with expression found in the lysosome making it a useful marker for activated phagocytic microglia (5). Galectin-3 has been shown to regulate inflammatory response in neurodegenerative diseases, released by microglia in response to inflammatory stimuli (6). Cathepsin B is a widely expressed cysteine peptidase located in the lysosome as well as processed and secreted, playing a role in microglial-mediated neuronal death (7). Hypoxia inducible factor-1 (HIF-1α) is a transcription factor responsible for adaptation to low oxygen environments whose downstream effects have been shown in a number of neurodegenerative diseases. Under normoxic conditions, HIF-1α is proline hydroxylated leading to ubiquitin mediated degradation (8). Axl is a receptor tyrosine kinase that binds Gas6, stimulating regulatory effects on microglial phagocytic response to inflammatory stimuli (9).

  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. Hopperton, K.E. et al. (2018) Mol Psychiatry 23, 177-98.
  6. Yip, P.K. et al. (2017) Sci Rep 7, 41689.
  7. Gan, L. et al. (2004) J Biol Chem 279, 5565-72.
  8. Zhang, Z. et al. (2011) Curr Med Chem 18, 4335-43.
  9. Grommes, C. et al. (2008) J Neuroimmune Pharmacol 3, 130-40.
Entrez-Gene Id
66824 , 558 , 1508 , 968 , 3958 , 3091 , 3059
Swiss-Prot Acc.
Q9EPB4 , P30530 , P07858 , P34810 , P17931 , Q16665 , P14317
For Research Use Only. Not For Use In Diagnostic Procedures.

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

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