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19171
Microglia Interferon-Related Module Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Microglia Interferon-Related Module Antibody Sampler Kit #19171

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Microglia Interferon-Related Module Antibody Sampler Kit: Image 1

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.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 2

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

Microglia Interferon-Related Module Antibody Sampler Kit: Image 3

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (10nM) for 30 min and Stat2 (D9J7L) 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 USP18, a known target gene of Stat2 (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 4

Flow cytometric analysis of U266 cells, untreated (blue) or treated with IFN-α (green) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 fragment (Alexa Fluor 488 Conjugate) #4412 was used as a secondary antibody.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of A172 (positive, left) or LNCaP (negative, right) cells using Akt3 (E1Z3W) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 6

Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY294002 #9901, Wortmannin #9951, and U0126 #9903 (50 μM, 1 μM, and 10 μM, 2 hr; blue) using Phospho-Akt (Ser473) (D9E) 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.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 7

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.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 8

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 9

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 10

Chromatin immunoprecipitations were performed with cross-linked chromatin from U266 cells treated with Human Interferon-α (IFN-α) #9906 (100 ng/ml) for 30 min, and either Stat2 (D9J7L) 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 USP18 promoter primers, SimpleChIP® Human WARS Intron 1 Primers #30101, 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.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 min; right) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (green) and β-actin (8H10D10) Mouse mAb #3700 (red).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 12

Western blot analysis of extracts from various cell lines and tissues using Akt3 (E1Z3W) Rabbit mAb (upper) and Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of C2C12 cells, LY294002-treated (left) or insulin-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5®#4084 (fluorescent DNA dye).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 14

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 15

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 16

Flow cytometric analysis of U266 cells using Stat2 (D9J7L) 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 647 Conjugate) was used as a secondary antibody.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 17

Western blot analysis of extracts from serum-starved U266 and A-431 cells, untreated (-) or treated with Human Interferon-α1 (hIFN-α1) #8927 (10 ng/ml; +) using Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb (upper) and total Stat2 (D9J7L) Rabbit mAb #72604 (lower).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 18

Western blot analysis of recombinant Akt1, Akt2, and Akt3 proteins using Akt3 (E1Z3W) Rabbit mAb (upper) and Akt1 (pan) (C67E7) Rabbit mAb #4691 (lower).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (left) or PTEN (138G6) Rabbit mAb #9559 (right). Note the presence of P-Akt staining in the PTEN deficient MDA-MB-468 cells.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 20

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 21

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

Microglia Interferon-Related Module Antibody Sampler Kit: Image 22

Confocal immunofluorescent analysis of A-431 cells, serum starved (left) or treated with IFNα (1000 U/ml for 30 mins; right) using Stat2 (D9J7L) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded human breast carcinoma comparing SignalStain® Antibody Diluent #8112 (left) to TBST/5% normal goat serum (right) using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 24

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).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 25

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

Microglia Interferon-Related Module Antibody Sampler Kit: Image 26

Immunoprecipitation of Stat2 from KARPAS-299 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Stat2 (D9J7L) Rabbit mAb. Western blot was performed using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 27

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 28

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

Microglia Interferon-Related Module Antibody Sampler Kit: Image 29

Western blot analysis of extracts from various cell lines using Stat2 (D9J7L) Rabbit mAb. KARPAS cell line source: Dr Abraham Karpas at the University of Cambridge.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 30

Immunohistochemical analysis using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb on SignalSlide® Phospho-Akt (Ser473) IHC Controls #8101 (paraffin-embedded LNCaP cells, untreated (left) or LY294002-treated (right)).

Microglia Interferon-Related Module Antibody Sampler Kit: Image 31

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded PTEN heterozygous mutant mouse endometrium using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. (Tissue section courtesy of Dr. Sabina Signoretti, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.)

Microglia Interferon-Related Module Antibody Sampler Kit: Image 33

Immunohistochemical analysis of paraffin-embedded U-87MG xenograft, untreated (left) or lambda phosphatase-treated (right), using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 34

Immunoprecipitation of phospho-Akt (Ser473) from Jurkat extracts treated with Calyculin A #9902 (100nM, 30 min). Lane 1 is 10% input, lane 2 is Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb, and lane 3 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900. Western blot analysis was performed with Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Microglia Interferon-Related Module Antibody Sampler Kit: Image 35

Western blot analysis of extracts from PC-3 cells, untreated or LY294002/wortmannin-treated, and NIH/3T3 cells, serum-starved or PDGF-treated, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb (upper) or Akt (pan) (C67E7) Rabbit mAb #4691 (lower).

To Purchase # 19171T
Product # Size Price Inventory
19171T
1 Kit  (6 x 20 µl)

Product Description

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

Specificity / Sensitivity

Each antibody in the Microglia Interferon-Related Module Antibody Sampler Kit detects endogenous levels of its target protein. Phospho-Stat2 (Tyr690) (D3P2P) Rabbit mAb recognizes endogenous levels of Stat2 protein only when phosphorylated at Tyr690. Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb detects endogenous levels of Akt only when phosphorylated at Ser473. 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.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Leu310 of mouse HS1, His140 of human Akt3, Leu706 of human Stat2, a phospho-specific synthetic peptide corresponding to residues surrounding Tyr690 of human Stat2 protein and Ser473 of human Akt, and recombinant mouse ASC/TMS1 protein.

Background

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).

Stat2 is critical to the transcriptional responses induced by type I interferons, IFN-alpha/beta (5,6). In response to IFN-alpha/beta, Stat2 is activated by phosphorylation at site Tyr690 through associations with receptor-bound Jak kinases (7). Akt is a protein kinase that plays a critical role in controlling survival and apoptosis. Akt is activated by various growth and survival factors to function in a wortmannin-sensitive pathway involving PI3 kinase (8-10) and its activity is shown to be essential for up-regulation of key IFN inducible proteins (11). Akt is activated by phospholipid binding and activation loop phosphorylation at Thr308 by PDK1 (12) and by phosphorylation within the carboxy terminus at Ser473. The previously elusive PDK2 responsible for phosphorylation of Akt at Ser473 has been identified as mammalian target of rapamycin (mTOR) in a rapamycin-insensitive complex with rictor and Sin1 (13,14).

  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. Fu, X.Y. et al. (1992) Proc Natl Acad Sci U S A 89, 7840-3.
  6. Ihle, J.N. (2001) Curr Opin Cell Biol 13, 211-7.
  7. Improta, T. et al. (1994) Proc Natl Acad Sci U S A 91, 4776-80.
  8. Franke, T.F. et al. (1997) Cell 88, 435-7.
  9. Burgering, B.M. and Coffer, P.J. (1995) Nature 376, 599-602.
  10. Franke, T.F. et al. (1995) Cell 81, 727-36.
  11. Kaur, S. et al. (2008) Proc Natl Acad Sci U S A 105, 4808-13.
  12. Alessi, D.R. et al. (1996) EMBO J 15, 6541-51.
  13. Sarbassov, D.D. et al. (2005) Science 307, 1098-101.
  14. Jacinto, E. et al. (2006) Cell 127, 125-37.

Pathways & Proteins

Explore pathways + proteins related to this product.

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