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. 2018 Feb 2;8(5):1286–1300. doi: 10.7150/thno.22552

Figure 4.

Figure 4

S6K1 binds and phosphorylates Mxi1 at Ser160 in vitro and in vivo. A. Endogenous S6K1 binds to Mxi1 and vice versa. HeLa cell lysates were subjected to immunoprecipitation using IgG, anti-Mxi1, or anti-S6K1 antibodies and then analyzed by Western blotting using indicated antibodies (n=3). B. HeLa cells stably expressing empty vector (EV), wild-type Mxi1, or Mxi1 S160A mutant were lysed and then analyzed by Western blotting using indicated antibodies (n=4). C. Lysates prepared from HeLa cells transfected with SFB-Mxi1 were treated with λ-PPase with or without λ-PPase inhibitor and then analyzed by Western blotting as indicated (n=3). D. Upper panel: His-Mxi1-WT or S160A protein were incubated in vitro with immunoprecipitates isolated from HEK293T cells transfected with HA-S6K1 construct and then analyzed by Western blotting using indicated antibodies. Lower panel: Proteins immunoprecipitated using anti-HA beads from HEK293T cells transfected with constructs encoding HA-S6K1 or HA-S6K1-KD were incubated with His-Mxi1-WT protein in vitro separately and then analyzed by Western blotting using indicated antibodies (n=3). E. A549 or H1299 cells were transfected with indicated S6K1 siRNA for 48 h and cell lysates were analyzed by Western blotting using antibodies as indicated (n=3). The ratio shows relative p-S160-Mxi1 or Mxi1 protein expression normalized for GAPDH (control, set at 1).