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9913
Phospho-p38 MAPK Pathway Sampler Kit
Primary Antibodies

Phospho-p38 MAPK Pathway Sampler Kit #9913

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Western Blotting Image 1

Western blot analysis of extracts from 293 cells, transfected with wt MSK1 or mutant MSK1, untreated or TPA-treated (200 nM), using Phospho-MSK1 (Thr581) Antibody.

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Western Blotting Image 2

Western blot analysis of extracts from COS and 293 cells, untreated or UV-treated, using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (upper) or p38 MAPK Antibody #9212 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated (-) or treated with TPA #4174 (200 nM, 15 min; +), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (upper), MKK3 (D4C3) Rabbit mAb #8535 (middle), or MKK6 (D31D1) Rabbit mAb #8550 (lower).

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Western Blotting Image 4

Western blot analysis of extracts from untreated or UV-treated COS cells, NIH/3T3 cells and C6 cells, using Phospho-ATF-2 (Thr71) (11G2) Rabbit mAb (upper), or ATF-2 (20F1) Rabbit mAb #9226 (lower).

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Western Blotting Image 5

Western blot analysis of extracts from HeLa or HT-29 cells, untreated (-) or treated (+) with either UV (40 mJ/cm2 with 30 min recovery) or anisomycin (25 μg/mL, 30 min), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from untreated or UV+TPA-treated HeLa and COS cells, using Phospho-MAPKAPK-2 (Thr334) (27B7) Rabbit mAb (upper), or MAPKAPK-2 Antibody #3042 (lower).

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Western Blotting 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.

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Western Blotting Image 8

Western blot analysis of extracts from 293 cells, untreated, UV-treated or calyculin A-treated, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 9

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

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IP Image 10

Immunoprecipitation of phospho-MKK3 (Ser189)/MKK6 (Ser207) from HeLa cells, untreated or treated with TPA #4174 (200 nM, 15 min), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (lanes 3 and 4) or Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lanes 5 and 6). Lanes 1 and 2 are 10% input. Western blot analysis was performed using

Phospho-MKK3 (Ser189)/MKK6 (Ser207) Antibody #9231. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used as a secondary antibody.

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Flow Cytometry Image 11

Flow cytometric analysis of THP-1 cells, untreated (blue) or Anisomycin treated (green), using Phospho-ATF-2 (Thr71) (11G2) Rabbit mAb compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 12

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, control (left) or λ phosphatase-treated (right), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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Western Blotting Image 13

Western blot analysis of extracts from 293 cells untreated or treated with UV and HeLa cells untreated or treated with calyculin A, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 14

Immunohistochemical analysis of paraffin-embedded mouse colon using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

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IF-IC Image 15

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or UV-treated (40 mJ/cm2 with 30 min recovery; right), using Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded mouse lung using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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IHC-P (paraffin) Image 17

Immunohistochemical analysis of paraffin-embedded human breast carcinoma, showing nuclear localization, using Phospho-MSK1 (Thr581) Antibody.

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded 293T cell pellets, untreated (left) or UV-treated (right), using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb.

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or UV-treated (right), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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Flow Cytometry Image 20

Flow cytometric analysis of Jurkat cells, untreated (blue) or anisomycin-treated (green), using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 21

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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IF-IC Image 22

Confocal immunofluorescent analysis of COS cells, untreated (left) or anisomycin-treated (right) using Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb (green). Actin filaments have been labeled with DY-554 phalloidin (red).

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IHC-P (paraffin) Image 23

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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Flow Cytometry Image 24

Flow cytometric analysis of HeLa cells, untreated (blue) or UV-treated (green), using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb.

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IF-IC Image 25

Confocal immunofluorescent analysis of C2C12 cells, untreated (left) or treated with λ phosphatase (middle), and NIH/3T3 cells (right) using Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). Negative staining in NIH/3T3 cells is in agreement with the observation that NIH/3T3 cells do not express HSP27 under basal conditions (5,7).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-MSK1 (Thr581) Antibody 9595 20 µl
  • WB
  • IP
  • IHC
H M 90 Rabbit 
Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb 4511 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk Mi Pg Sc 43 Rabbit IgG
Phospho-MKK3 (Ser189)/MKK6 (Ser207) (D8E9) Rabbit mAb 12280 20 µl
  • WB
  • IP
  • IF
H M R Mk 38 kDa MKK6, 40 kDa MKK3 Rabbit IgG
Phospho-ATF-2 (Thr71) (11G2) Rabbit mAb 5112 20 µl
  • WB
  • F
H M R Mk 70 Rabbit IgG
Phospho-HSP27 (Ser82) (D1H2F6) XP® Rabbit mAb 9709 20 µl
  • WB
  • IHC
  • IF
  • F
H M 27 Rabbit IgG
Phospho-MAPKAPK-2 (Thr334) (27B7) Rabbit mAb 3007 20 µl
  • WB
H M R Mk 49 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

The Phospho-p38 MAPK Pathway Sampler Kit provides an economical means to evaluate the activation status of multiple members of the p38 MAPK pathway, including phosphorylated MSK1, p38 MAPK, MKK3/MKK6, ATF-2, HSP27 and MAPKAPK-2. The kit includes enough primary and secondary antibodies to perform two Western blot experiments.

Each antibody in the Phospho-p38 MAPK Pathway Sampler Kit recognizes only the phosphorylated form of its specific target.

Monoclonal antibodies are produced by immunizing animals with synthetic phosphopeptides corresponding to residues surrounding Thr581 of human MSK1, Thr180/Tyr182 of human p38 MAPK, Ser189 of human MKK3, Thr71 of human ATF-2, Ser82 of human HSP27 or Thr334 of human MAPKAPK-2.

p38 MAP kinase (MAPK), also called RK (1) or CSBP (2), is the mammalian orthologue of the yeast HOG kinase that participates in a signaling cascade controlling cellular responses to cytokines and stress (1-4). Four isoforms of p38 MAPK, p38α, β, γ (also known as Erk6 or SAPK3), and δ (also known as SAPK4) have been identified. Similar to the SAPK/JNK pathway, p38 MAPK is activated by a variety of cellular stresses including osmotic shock, inflammatory cytokines, lipopolysaccharide (LPS), UV light, and growth factors (1-5). MKK3, MKK6, and SEK activate p38 MAPK by phosphorylation at Thr180 and Tyr182. Activated p38 MAPK has been shown to phosphorylate and activate MAPKAP kinase 2 (3) and to phosphorylate the transcription factors ATF-2 (5), Max (6), and MEF2 (5-8). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole) is a selective inhibitor of p38 MAPK. This compound inhibits the activation of MAPKAPK-2 by p38 MAPK and subsequent phosphorylation of HSP27 (9). SB203580 inhibits p38 MAPK catalytic activity by binding to the ATP-binding pocket, but does not inhibit phosphorylation of p38 MAPK by upstream kinases (10).

Four residues (Thr25, Thr222, Ser272 and Thr334) of MAPKAPK-2 are phosphorylated by p38 in an in vitro kinase assay (3). Phosphorylation at Thr222, Ser272 and Thr334 seems to be essential for the activity of MAPKAPK-2 (3). Activated MAPKAPK-2 can in return phosphorylate HSP27 at serines 15, 78 and 82 (3,9). Phosphorylation of HSP27 causes a change in the tertiary structure of HSP27, which shifts from large homotypic multimers to dimmers and monomers (10). It has been illustrated that phosphorylation and increased concentration of HSP27 modulate actin polymerization and reorganization (11,12).

  1. Rouse, J. et al. (1994) Cell 78, 1027-37.
  2. Han, J. et al. (1994) Science 265, 808-11.
  3. Lee, J.C. et al. (1994) Nature 372, 739-46.
  4. Freshney, N.W. et al. (1994) Cell 78, 1039-49.
  5. Raingeaud, J. et al. (1995) J Biol Chem 270, 7420-6.
  6. Zervos, A.S. et al. (1995) Proc Natl Acad Sci U S A 92, 10531-4.
  7. Zhao, M. et al. (1999) Mol Cell Biol 19, 21-30.
  8. Yang, S.H. et al. (1999) Mol Cell Biol 19, 4028-38.
  9. Cuenda, A. et al. (1995) FEBS Lett 364, 229-33.
  10. Kumar, S. et al. (1999) Biochem Biophys Res Commun 263, 825-31.
  11. Landry, J. et al. (1992) J. Biol. Chem. 267, 794-803.
  12. Rogalla, T. et al. (1999) J. Biol. Chem. 274, 18947-18956.
  13. Lavoie, J. et al. (1993) J. Biol. Chem. 268, 24210-24214.
  14. Rousseau, S. et al. (1997) Oncogene 15, 2169-2177.
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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