Figure 2.
Figure 2. Smurf1 mediates Kindlin-2 proteasomal degradation. (A) GFP-Kindlin-2 plasmid (2 µg) was transfected into HEK293T cells together with increasing amounts of Smurf1 WT plasmid. Kindlin-2 expression was determined by immunoblotting with an anti-GFP antibody 24 h after transfection. (B) HEK293T cells were transfected with increasing amounts of Smurf1 expression plasmid, and endogenous protein levels of Kindlin-2 were determined. (C) GFP-Kindlin-2 was transfected into HEK293T cells together with Smurf1 WT or C699A plasmid, and Kindlin-2 expression was examined. (D) HEK293T cells with transfection of GFP-Kindlin-2 and Flag-Smurf1 plasmids were treated with a proteasome inhibitor MG132 (20 µM) or DMSO for 6 h. Kindlin-2 expression was measured. (E) HEK293T cells were transfected with control siRNA or Smurf1 siRNA, and the expression of Kindlin-2 was detected. (F) Flag-Smurf1 was transfected into HeLa cells together with GFP-Kindlin-2, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of GFP-Kindlin-2 was measured by Western blot. Quantification of the Kindlin-2 half-life was performed, and each point is represented as the mean ± SD of triplicate experiments. (G) HeLa cells were transfected with Flag-Smurf1, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of endogenous Kindlin-2 protein was measured by Western blot and analyzed. Each point represents the mean ± SD of triplicate experiments. (H) CHX-chase experiments of Kindlin-2 in MDA-MB-231 cells transfected with control siRNA or Smurf1 siRNA are shown. Quantification of Kindlin-2 half-life was performed, and each point is represented as the mean ± SD (n = 3). Refer to the image caption for details.

Smurf1 mediates Kindlin-2 proteasomal degradation. (A) GFP-Kindlin-2 plasmid (2 µg) was transfected into HEK293T cells together with increasing amounts of Smurf1 WT plasmid. Kindlin-2 expression was determined by immunoblotting with an anti-GFP antibody 24 h after transfection. (B) HEK293T cells were transfected with increasing amounts of Smurf1 expression plasmid, and endogenous protein levels of Kindlin-2 were determined. (C) GFP-Kindlin-2 was transfected into HEK293T cells together with Smurf1 WT or C699A plasmid, and Kindlin-2 expression was examined. (D) HEK293T cells with transfection of GFP-Kindlin-2 and Flag-Smurf1 plasmids were treated with a proteasome inhibitor MG132 (20 µM) or DMSO for 6 h. Kindlin-2 expression was measured. (E) HEK293T cells were transfected with control siRNA or Smurf1 siRNA, and the expression of Kindlin-2 was detected. (F) Flag-Smurf1 was transfected into HeLa cells together with GFP-Kindlin-2, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of GFP-Kindlin-2 was measured by Western blot. Quantification of the Kindlin-2 half-life was performed, and each point is represented as the mean ± SD of triplicate experiments. (G) HeLa cells were transfected with Flag-Smurf1, and cells were treated with CHX at 100 µg/ml for the indicated times. The half-life of endogenous Kindlin-2 protein was measured by Western blot and analyzed. Each point represents the mean ± SD of triplicate experiments. (H) CHX-chase experiments of Kindlin-2 in MDA-MB-231 cells transfected with control siRNA or Smurf1 siRNA are shown. Quantification of Kindlin-2 half-life was performed, and each point is represented as the mean ± SD (n = 3).

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