Fig. 5. Kindlin-2 increases Foxo1 protein stability and polyubiquitination.

a Western blotting. Protein extracts from livers of control and Kindlin-2 Het mice were subjected to western blotting using the indicated antibodies (n = 2). b, c shRNA Kindlin-2 KD. Lentivirus expressing control shRNA (sh-NC) or Kindlin-2 shRNA (sh-K2) were used to infect Huh7 or HepG2 cells, followed by western blotting (b) and qRT-PCR analyses (c) (n = 3) to determine the expression of Foxo1 and Kindlin-2 protein and mRNA, respectively. d–g Cycloheximide (CHX) experiments. HepG2 cells (d, e) or Huh7 cells (f, g) with and without Kindlin-2 shRNA KD were treated with 100 μg/mL of CHX for the indicated times, followed by western blotting for expression of Kindlin-2. h Kindlin-2 overexpression (OE) reduces Foxo1 ubiquitination. HEK293T cells were transiently transfected with V5-tagged Foxo1 plasmid with and without Flag-tagged Kindlin-2 plasmid. At 48 h after the transfection, cells were pretreated with or without MG132 (10 μM) for 6 h, followed by immunoprecipitation (IP) and immunoblotting (IB) with the indicated antibodies. i, j Kindlin-2 KD increases endogenous Foxo1 polyubiquitination. HepG2 cells and Huh7 cells were transfected with lentivirus-expressing control shRNAs or Kindlin-2-specific shRNAs. The cells were pretreated with MG132 (10 μM) for 6 h, followed by IP and IB assays with the indicated antibodies. For h–j, 200 μg of whole-cell extracts from the sh-NC group and 800 μg of whole-cell extracts from the sh-K2 group were used for the IP assays. Immunoprecipitates were resuspended in 50 μl buffer. Fifteen microlitres from each sample were loaded for SDS-PAGE, followed by western blotting analyses. Protein extracts (20 μg) were used for western blotting from each sample for (a, b, d, f, h–j) (input panels). c, e, g Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, determined by two-tailed Student’s t-test. b, d, f, h–j Data are representative of three biologically independent replicates.