Fig. 5: Pim1 promotes the Akt-signaling pathway by directly phosphorylating TRAF6.

a WT and Pim1^−/− BMMs were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml), and total cell lysates were collected at the indicated times and subjected to immunoblotting with the indicated antibodies. Band intensity was quantified with ImageJ. Left: representative immunoblot. Right: plots showing the mean phospho-Akt/total Akt ratio (top) and phospho-GSK3β/total GSK3β ratio (bottom). b, c HEK 293T cells were transfected with the indicated combinations of expression plasmids, followed by co-immunoprecipitation with an anti-Flag antibody. The proteins in the immunoprecipitates were detected by immunoblotting with the indicated antibodies. The expression levels of the transfected plasmids were verified by immunoblot analysis of the whole-cell lysates. Representative immunoblots are shown. d Cell lysates from mature WT and Pim1^−/− osteoclasts were subjected to immunoprecipitation with an anti-TRAF6 antibody. The proteins in the total cell lysates were detected by immunoblotting with the indicated antibodies. Band intensity was quantified with ImageJ. Left: a representative immunoblot. Middle: plot showing the average phospho-Akt/total AKT ratios. Right: plot showing the average serine-phosphorylated TRAF6 levels relative to total TRAF6 levels. All data are representative of three independent experiments. The data are presented as mean ± s.d. Statistical analysis was conducted with two-tailed unpaired Student’s t-test and two-way ANOVA with Tukey’s multiple comparison test. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.