Fig 1: DAPK1-deficiency selectively inhibits phosphorylation of RIPK1(S321), MK2, and p38 MAPK.a DAPK1-deficiency does not affect MKK3 activation. WT and Dapk1-/- BMDMs were treated with TNF, and levels of pMKK3 and MKK3 were determined in cell lysates. b DAPK1[K42A] increases resistance to necroptosis in DAPK1-deficient cells. DAPK1-/- HT-29 cells were transfected with empty vector (EV), or transfected with WT DAPK1 or DAPK1[K42A]. Cells were treated with zVAD (Ctrl) or zVAD + BV6, and viability determined by ATP assay. c Either WT DAPK or DAPK1[K42A] increases p38 MAPK activation in DAPK1-null cells. DAPK1-/- HT-29 cells, mock transduced, transduced with WT DAPK1 or DAPK1[K42A] were treated with TNF (20 ng/ml) for the indicated time points, and the levels of DAPK1, p38 MAPK and phospho-p38 MAPK were determined. d Overexpression of p38 MAPK inhibits necroptosis. DAPK1-/- HT-29 cells were transfected with empty vector (EV), p38 MAPK or MKK3. The extent of necroptosis induced by zVAD+BV6 was determined by ATP release. e Interaction between DAPK1 and p38 MAPK. HEK293T cells were transfected with DAPK1-FLAG and/or p38-HA as indicated. Cell lysates were prepared 24 later, and precipitated with anti-FLAG or anti-HA. f p38 MAPK binds DAPK in vitro. Recombinant human p38 MAPK (200 ng) was incubated with purified DAPK (100 ng), as indicated. p38 MAPK was pulled down by anti-p38 MAPK (ab170099, Abcam), and the presence of DAPK and p38 MAPK in immunoprecipitants determined by anti-FLAG and anti-His, respectively. Results have been independently confirmed using another anti-p38 MAPK (7218, Cell Signaling). g DAPK1 promotes MKK3-directed p38 MAPK phosphorylation. Recombinant p38 MAPK (50 ng), MKK3 (100 ng), and DAPK1-FLAG (25 ng or 75 ng) was incubated as indicated in ATP-containing kinase buffer at 30 °C for 1 h. The amounts of p38 MAPK, MKK3, DAPK1 and phospho-p38 MAPK was determined by immunoblots. h Binding of different DAPK1 mutants to p38 MAPK. HEK293T cells were transfected with different mutants of DAPK1-FLAG and p38-HA as indicated. The presence of p38-HA in anti-FLAG precipitates was determined. i Failure of DAPK1(?Cyt) to increase p38 MAPK activation. DAPK1, DAPK1(?Cyt) or DAPK1(?CAM) was evaluated for its ability to increase MKK3-directed p38 MAPK activation described in (g). Values are mean ± SD of triplicates in a single experiment. *P < 0.05, ***P < 0.001 (b, d) for two-way ANOVA followed by a Tukey’s multiple comparison test. Results have been repeated in three (a–c, e, f) or two (d, g–i) independent experiments.
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