Figure 5. CSN6 phosphorylation mutant S60A has different stability. (A) The steady-state expression of phosphorylation mutant CSN6S60A. 293T cells were co-transfected with indicated plasmids and increasing amounts of Akt. Equal amounts of cell lysates were immunoblotted with indicated antibodies. (B) Turnover rate of CSN6 phosphorylation mutant S60A is accelerated. Indicated wt and CSN6 mutant tranfected cells were treated with cycloheximide (CHX) (100 µg/ml) for the indicated times. Cell lysates were immunoblotted with indicated antibodies. (C) Akt upregulates CSN6 by decreasing CSN6 ubiquitination. 293T cells were transfected with the indicated plasmids. The proteasome inhibitor MG132 was added 6 h before harvesting cells. The ubiquitinated amount of CSN6 is analyzed by immunoprecipitated (IP) with anti-myc followed by immunoblotting (IB) with anti-HA. (D) Akt reduces CSN6 ubiquitination in the presence of FBXW7. 293T cells were transfected with the indicated plasmids. The proteasome inhibitor MG132 was added 6 h before harvesting cells. The ubiquitinated amount of CSN6 is analyzed by immunoprecipitated (IP) with anti-GFP followed by immunoblotting (IB) with anti-ubi.