Fig. 5: CBL-b E3 ligase activity at C373 promotes PARP-1 neddylation in Pi-induced VC.

a PARP-1 neddylation in Pi-treated A10 cells is dependent on CBL-b E3 ligase activity. b PARP-1 activity was determined via an anti-PARP-1 antibody. Mutants with inactive CBL-b E3 ligase activity showed decreased PARP-1 activity in Pi-treated A10 cells. n = 4 per group, independent experiments. c Measurement of the calcium content. Pi increased calcium accumulation, but this effect was blocked by inactive CBL-b E3 ligase mutants (C373A and W400A). n = 11 per group, independent experiments. d Alizarin red S staining was performed. Scale bar, 10 mm. e PLA assay. NEDD8 was associated with PARP-1 under Pi conditions, but was dissociated by the CBL-b C373 peptide. Scale bar, 20 µM. f Immunoblot analysis was performed via immunoprecipitation with anti-PARP-1. g A boronate pull-down assay was performed to detect the PAR polymer. h PARP-1 activity was analyzed. Pi-induced PARP-1 activity was attenuated by CBL-b C373. n = 4 per group, independent experiments. i Calcium assay. CBL-b C373 inhibited Pi-induced calcium deposition in A10 cells. n = 6 per group, independent experiments. j Calcification was determined by alizarin red S staining. Scale bar, 10 mm. k Quantification of alizarin red S staining was performed. n = 6 per group, independent experiments. “*” indicates a nonspecific band. The data are presented as the means ± SEMs. Statistical significance was tested via ANOVA with Tukey’s HSD test and Dunnett’s T3 test.