Fig. 5: March5 inhibits Trim28-mediated ubiquitination of Kindlin-2 by directly ubiquitinating Trim28.

a Western blot analyses for March5, Trim28, and actin expression in isolated islets from 10-week-old male cKO and CON mice. b, c Western blot analyses. INS1 cells were co-transfected with siMarch5 plasmids for 48 h, the protein extracts were then subjected to western blot analyses with the indicated antibodies. N = 3 biologically independent samples (a–c). d QPCR analyses. Total RNA isolated from the primary islets of 2-month-old male cKO and CON mice was subjected to qPCR for Trim28 mRNA expression. N = 3 per group. Trim28 mRNA was normalized to Gapdh mRNA. e QPCR analyses. Total RNA isolated from INS1 cells, as treated in (b), was subjected to qPCR for Trim28 expression. All mRNA levels were normalized to Gapdh mRNA. N = 3 biologically independent samples. f, g CHX experiments. HEK293T cells with or without March5 knockdown were treated with 100 μg/mL of CHX for the indicated times, followed by western blot analyses for expression of Trim28. Quantitative data from three independent experiments (g). h, i Western blot analyses. Effect of MG132 on Trim28 protein expression in INS1 cells. j March5 knockdown decreases endogenous Trim28 ubiquitination. k March5 overexpression increases endogenous Trim28 ubiquitination. l Loss of function of March5 (H43W) decreases Trim28 ubiquitination. N = 3 biologically independent samples (h–l). m, n CHX experiments. HEK293T cells with or without Trim28 knockdown were treated with 100 μg/mL of CHX for the indicated times, followed by western blot analyses for Kindlin-2 expression. Quantitative data from three independent experiments (n). o Trim28 knockdown decreases endogenous Kindlin-2 ubiquitination. HEK293T cells were transiently transfected with control siRNAs or siTrim28. At 48 h post-transfection, cells were treated with MG132 (10 μM) for 6 h, then IP and IB with the indicated antibodies were performed. p Trim28 knockdown abrogates the increased endogenous Kindlin-2 ubiquitination by March5 deficiency. q Trim28 overexpression restores the decreased endogenous Kindlin-2 ubiquitination by March5 overexpression. N = 3 biologically independent samples (o–q). Data are expressed as mean ± s.d. Data were analyzed by two-tailed unpaired Student’s t-tests for comparisons between two groups and one-way ANOVA for multiple groups, followed by Tukey’s post hoc test. Source data are provided as a Source Data file.