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. 2018 Jan 19;9(11):9808–9824. doi: 10.18632/oncotarget.24275

Figure 6. RRD-251 promotes D3-induced nuclear localization and regulation of c-Raf.

Figure 6

Changes in total expression and/or the specific phosphorylation of c-Raf were assessed by Western blotting. 25 μg of protein from cytoplasmic and nuclear lysates collected 48 hours after treatment were resolved on 12% polyacrylamide gels. (A-B) D3 induces the nuclear translocation of c-Raf concurrent with a small increase in cytoplasmic expression, effects which are augmented with RRD-251 co-treatment. (C-D) D3 promotes the phosphorylation of c-Raf at serine 259 in both cytoplasmic and nuclear samples; addition of RRD-251 enhances phosphorylation at this site in both compartments. (E-F) D3 promotes the phosphorylation of c-Raf at serine 621 in the cytoplasm, and RRD-251 enhances this in co-treated samples. Nuclear phosphorylation of c-Raf at serine 621 is slightly decreased, with no enhancement in co-treated samples. (G-H) D3 promotes the phosphorylation of the p-c-Raf CTD (ser 289/296/301) in both the cytoplasm and nucleus, phosphorylation of the c-terminal domain is augmented by RRD-251 co-treatment. (I-J) Loading control used: GAPDH for cytosolic proteins and TBP for nuclear proteins.