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. 2015 Mar 10;25(5):561–573. doi: 10.1038/cr.2015.30

Figure 6.

Figure 6

ERK destabilizes FBW7 by promoting its ubiquitination. (A) Immunoblot (IB) analysis of whole-cell lysates (WCL) and anti-Flag immunoprecipitates of 293T cells transfected with HA-ERK1 together with Myc-Ub and various Flag-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (B) Immunoblot (IB) analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with wild-type HA-ERK1 or kinase-dead (KD) mutant together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (C) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of Hela cells transfected with Flag-WT-FBW7 together with Myc-Ub and siRNA against ERK1/2. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (D) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and Myc-Ub constructs. Thirty hours posttransfection, cells were treated with MG132 and trametinib (where indicated) overnight before harvest. (E) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with HA-KRASG12D together with Myc-Ub and Flag-WT-FBW7 constructs. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest. (F) Immunoblot analysis of WCL and anti-Flag immunoprecipitates of 293T cells transfected with Flag-WT-FBW7 and HA-ERK1 together with Myc-Ub and siRNA oligos against Pin1. Thirty hours posttransfection, cells were treated with MG132 overnight before harvest.