Smurf1 induces polyubiquitination of Kindlin-2. (A) Flag-Kindin-2 and HA-Ub were cotransfected into HEK293T cells together with control vector, Myc-Smurf1 (WT), or Myc-Smurf1 (C699A) expression plasmid. Kindlin-2 ubiquitination was detected by immunoprecipitation with anti-Flag M2 beads and immunoblotting with an anti-HA antibody. (B) E1, UbcH5c (E2), HA-Ub, GST-Smurf1 (expressed and purified from bacteria), and His-Kindlin-2 (expressed and purified from bacteria) were incubated at 30°C for 2 h in ubiquitination reaction buffer. Ubiquitinated Kindlin-2 was visualized by immunoblotting with an anti-HA antibody. (C) MDA-MB-231 cells were transfected with indicated siRNAs for 48 h and pretreated with proteasome inhibitor MG132 (10 µM) for 12 h. Polyubiquitination of endogenous Kindlin-2 was detected by anti-ubiquitin antibody. (D) HEK293T cells were transfected with Flag-Kindlin-2, Myc-Smurf1, and various Ub mutant plasmids. 24 h after transfection, an in vivo ubiquitination assay was performed. (E) HA-Ub WT or mutants of K27 and K33 were transfected into HEK293 cells together with Flag-Kindlin-2 and Myc-Smurf1 or control vector. Kindlin-2 ubiquitination was detected. (F) HEK293T cells were transfected with HA-Ub, Myc-Smurf1, and various Kindlin-2 mutant plasmids, and an in vivo ubiquitination assay was performed. (G) HEK293T cells were transfected with Myc-Smurf1 and Kindlin-2 mutant plasmid, and Kindlin-2 levels were assessed. (H) Myc-Smurf1 was transfected into HeLa cells together with Flag-Kindlin-2 WT or Flag-Kindlin-2 K153/154+187R mutant, and cells were treated with 100 µg/ml CHX for the indicated times. The half-life of Flag-Kindlin-2 was measured by Western blot. Quantification of Kindlin-2 half-life was performed, and each point is represented as mean ± SD of triplicate experiments.