Figure 3: HGK induces the lysosomal degradation of TRAF2.

(a) Immunoblotting analyses of HGK and TRAFs in primary splenic T cells of WT and T-HGK cKO mice. (b) Flow cytometry analyses of TRAF2-positive cells from WT and HGK cKO (cKO) T cells transfected with empty vector (Vec, encoding CFP alone) or vector encoding HGK tagged with CFP. Means±s.e.m. are shown. (c) Immunoblotting analyses of TRAF2 and actin in Jurkat T cells stimulated with anti-CD3 antibodies (upper panel). In vitro kinase assays of the endogenous HGK proteins immunoprecipitated from lysates of Jurkat T cells stimulated with anti-CD3 antibodies (lower panel). NS, normal serum. n=3. Means±s.e.m. are shown. (d) Immunoblotting analyses of indicated molecules in lysates of HEK293T cells transfected with TRAF2 (2 μg) and HGK (various amounts) plasmids. (e) Immunoblotting analyses of indicated molecules in lysates of HEK293T cells transfected with TRAF2 and/or HGK, and treated with the lysosome inhibitor chloroquine. (f) Confocal microscopy analyses of co-localization of HGK-CFP (blue), TRAF2-YFP (green) and lysosome (lysotracker; red) in HEK293T cells treated with chloroquine. Original magnification, × 630; scale bar, 10 μm. Data shown are representatives of three independent experiments.