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. 2020 Mar 24;11(12):1017–1036. doi: 10.18632/oncotarget.27508

Figure 1. Roscovitine enhances the amount of ATRA-induced phosphorylated c-Raf and phosphorylated c-Raf in the nucleus modulates the RB protein functions.

Figure 1

(A–C) Western blot of c-Raf and its phospho-regulatory residues in HL-60 cells cultured with ATRA for 72 h showed that ATRA upregulated nuclear c-Raf, pS259 and pS289/296/301 c-Raf expression and co-treatment with ATRA plus roscovitine further increased of c-Raf and its active phosphorylation sites, pS259 and pS289/296/301, compared to ATRA alone. (D) TATA binding protein (TBP) is the loading control. (E) c-Raf immunoprecipitates probed for RB or S608 RB show that roscovitine enhances ATRA-induced downregulation of the amount of nuclear c-Raf complexed with RB and specifically with its serine 608 phosphorylated form (pS608 RB). An equal amount of pre-cleared nuclear lysate was collected 72 h post treatment and incubated overnight with 2.5 μg of the precipitating antibody with magnetic beads and resolved on 12 % polyacrylamide gels. All blots shown are representative of three replicates.