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. Author manuscript; available in PMC: 2019 Jun 16.
Published in final edited form as: Mol Cell. 2017 Jul 6;67(3):484–497.e5. doi: 10.1016/j.molcel.2017.06.011

Figure 1. Akt is a Bcl3 kinase.

Figure 1.

(A) MS/MS spectrum (left) showing phosphorylation of Bcl3 Ser33 and Ser446; fragment ion mass table (right) of the phosphopeptide. (B) In vitro kinase assay showing recombinant Akt phosphorylate Bcl3 wt and S446E, but not S33E. Commassie staining showing Bcl3 protein levels used for the kinase assay. (C) WB analysis of nuclear and cytoplasmic extracts of RAW 264.7 cells after LPS stimulation for different times. Both wt and p-Ser33 Bcl3 accumulated in the nucleus in LPS treated cells. (D) Akt Inhibitor VIII treatment decreased both the total amounts of endogenous Bcl3 as well as the p-Ser33 Bcl3 in LPS treated RAW 264.7 cells. (E) Wortmannin and Akt Inhibitor VIII inhibited Bcl3 transcriptional activity with p52 on P-Selectin reporter. (F) Luciferase assay showing p52:Bcl3 transcriptional activity was significantly decreased in Akt2 KD HeLa cells. The data was analyzed from three independent experiments performed in triplicate. RLU (Relative Luciferase Unit) *p<0.05, **p<0.01. Error bars represent standard deviation (SD).