Fig. 6

The interaction between Kremen2 and SOCS3 inhibits EGFR ubiquitination and degradation. A HEK293T cells were transfected with the Flag-Krm2 plasmid. IB analysis of exogenous Kremen2, EGFR, AKT, STAT3, and SOCS3, as assessed after co-IP with IgG or anti-Flag. B Co-localization of red (SOCS3) and green (Kremen2) was analyzed by Image-Pro Plus 6.0. C HEK293T cells were transfected with His-SOCS3 and Flag–Krm2. Ni–NTA beads were used to pull down His-tagged SOCS3, and immunoblotted with the indicated antibodies. D EGFR expression levels were measured in A549 cells and Kremen2 over-expressing A549 cells transfected with the vector or His-SOCS3 plasmid. E A549 cells expressing the vector or His-SOCS3 were treated with MG132 or not. F A549 cells stably expressing the vector or His-SOCS3 were treated with CHX (0.1 mg/ml) and collected at the indicated time points. The intensity of EGFR bands in western blot was measured with ImageJ software and normalized to the corresponding GAPDH and plotted. G, H IB analysis of the endogenous EGFR and SOCS3 in H1703 cells, as assessed after co-IP with anti–EGFR or anti-SOCS3. I A549 cells transfected with the indicated plasmids were treated with 20 μM MG132 for 8 h before co-IP. J HEK293T cells were transfected with indicated plasmids and treated with 20 μM MG132 for 8 h. Ni–NTA beads were used to pull down His-tagged EGFR, and the polyubiquitylated EGFR protein was examined