Figure 3.

Activation of p38^MAPK results in phosphorylation of SRC-3 at S857. (A) p38^MAPK but not ERK1/2 is involved in phosphorylation of SRC-3 at S857. H1299 cells were incubated with either 10 μM MEK1/2 inhibitor (PD-184352) or 10 μM p38^MAPK inhibitor (SB-202190) for 2 h before SRC-3 was immunoprecipitated (IP). The IP lysate and whole cell extract (WCE) were analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho ERK1/2 MAPK and anti-ERK2 antibodies. (B–F) p38^MAPK activation phosphorylates SRC-3 at S857. The full-length blots are presented in supplementary figure S12. H1299 (B), A549 (C), HEK 293 (D), HeLa (E) and MDA MB 231 (F) cells were stimulated with either 10 ng/ml TNF-α (15 min), 10 μg/ml anisomycin or 250 μM sodium arsenite (SA) for 30 min. Unstimulated cells were used as control. The cells were lysed and the level of phosphorylation of SRC-3 at S857 and p38^MAPK at T180/Y182 was analyzed by Western-blotting using anti-P-S857-SRC-3, anti-SRC-3, anti-phospho-p38^MAPK and anti-p38^MAPK antibodies. The full-length blots are presented in supplementary figures S13–S17. (G,H) Inhibition of p38^MAPK activation prevents TNF-α and anisomycin-induced phosphorylation of SRC-3 at S857. A549 cells were seeded and left overnight. On the other day, the cells were pretreated either with DMSO or 10 μM SB-202190 for 30 min. Then they were stimulated with 10 ng/ml TNF-α (15 min) or 10 μg/ml anisomycin (G) or 500 μM sodium arsenite (SA) (H) for 30 min. Finally, the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total amount of SRC-3, HSP27 and actin were detected by Western-blotting using appropriate antibodies. The full-length blots are presented in supplementary figures S18,S19.