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. 2011 Jun 20;108(27):11127–11132. doi: 10.1073/pnas.1104128108

Fig. 4.

Fig. 4.

Msn kinases directly phosphorylate α-helix 1 of Smad. (A) HA-Msn and FLAG-MAD were expressed in S2 cells. After OA stimulation, FLAG-MAD was immunoprecipitated and evaluated by immunoblot analysis. (B) 293T cells were transfected and treated with OA as indicated. FLAG-Smad1 was immoprecipitated, and phosphorylation on T322 was measured by immunoblot analysis. (C) IP-kinase assays. HA-tagged WT and catalytic mutants of MAP4K4, MINK1, and TNIK (KM, K54R mutation in all three cases) were purified from 293T cells with or without OA treatment as indicated. FLAG-Smad1 was affinity-purified separately and used as the substrate. (D) Recombinant MINK1 kinase domain (GST-MINK1K) purified from E. coli directly phosphorylated purified recombinant Smads in vitro.