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. 2020 Jul 9;10:11388. doi: 10.1038/s41598-020-68219-4

Figure 4.

Figure 4

MK2 phosphorylates SRC-3 at S857. (A,B) MK2 phosphorylates SRC-3 at S857 in mouse cell lines. Mouse embryonic fibroblast (MEF) cells (A) or bone marrow derived dendritic cells (BMDC) cells (B) derived from mice knocked out for MK2 and MK3 expression (MK2/MK3−/−), rescued with retroviral transduced GFP-MK2 wild type (MK2/MK3−/− + MK2WT), or rescued with retroviral transduced kinase dead MK2 (MK2/MK3−/− + MK2K72A) were seeded and left overnight. Then the cells were treated with either 10 ng/ml TNF-α, 250 μM SA or 5 ng/ml Lipopolysaccharide (LPS) as indicated for 30 min. Cell lysates were analyzed by Western-blotting using anit-P-S857-SRC-3, anti-SRC-3, anti-MK2 and anti-actin antibodies. *indicates the phosphorylated band. The full-length blots are presented in supplementary figures S20,S21. (C,D) PF-3644022 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 with DMSO or 1, 2.5, 5, 10 μM of PF-3644022 for 30 min followed by stimulation with either 10 ng/ml TNF-α (C) or 10 μg/ml anisomycin (D) for 30 min. Then the cells were lysed and level of phosphorylation of SRC-3 at S857, HSP27 at S82, total SRC-3 and total HSP27 were analyzed by Western-blotting using anti-PS857-SRC-3, anti-PS82-HSP27, anti-SRC-3 and anti-HSP27 antibodies respectively. The full-length blots are presented in supplementary figures S22,S23. (E) MK2 phosphorylates SRC-3 at S857 in the human lung adenocarcinoma cancer cell line A549. A549 cells were seeded in a 6-well plate and left overnight then co-transfected with 20 nM siRNA against SRC-3 and 1 μg vector expressing either siRNA resistant SRC-3 wild type-FLAG (SRC-3 WT-FLAG) or siRNA resistant SRC-3 S857A-FLAG (SRC-3 S857A-FLAG). After 48 h, cells were treated with 10 ng/ml TNF-α (15 min) or 10 μM anisomycin for 30 min in absence or presence of 10 μM PF-3644022, which was added 30 min before the treatment. The cells were lysed and phosphorylation of SRC-3 at S857 was investigated by Western-blotting using anit-P-S857-SRC-3, anti-FLAG and anti-actin antibodies. The full-length blots are presented in supplementary figure S24. (F–J) MK2 phosphorylates endogenous SRC-3 at S857 in human cell lines. HeLa (F), A594 (G), H1299 (H), HEK 293 (I) and MDA MB 231 (J) cells were seeded and left overnight. Then the cells were treated with 10 μM PF-3644022 for 30 min followed by stimulation with 10 μg/ml anisomycin for 30 min. Cells were lysed and level of phosphorylation of SRC-3-S857 and p38MAPK were detected, before the total amount of SRC-3, p38MAPK and actin were detected by Western-blotting using appropriate antibodies. Presented here is a representative image of three independent experiments that showed similar result. The full-length blots are presented in supplementary figure S25–S29.