Figure 1

ROS generation and apoptosis are increased in CD2AP−/− podocytes. (A) ROS production is increased in the absence of CD2AP (CD2AP−/−) compared to wild type (WT) podocytes as observed by DCFH-DA fluorescent probe assay. Hoechst 33342 was used for normalization. (B) Flow cytometry with annexin V and 7-AAD double labelling indicates that absence of CD2AP induces apoptosis. Reintroduction of CD2AP into CD2AP−/− podocytes rescues CD2AP−/− podocytes from apoptosis. (C) Representative immunoblot of CD2AP expression in WT podocytes, and in CD2AP−/− podocytes infected with lentiviruses containing an empty vector (EV) or human CD2AP cDNA. (D,E) Immunoblotting and quantification reveals that phosphorylation of AKT on T308 (p-T308) is reduced in CD2AP−/− podocytes compared to WT podocytes. Total AKT (panAKT) is used for normalization. Full width blot of (D) is shown in Supplemental Fig. S1A. (F) In-cell Western and quantification shows no difference in the phosphorylation of AKT on S473 (p-S473) in relation to total AKT (panAKT) between WT and CD2AP−/− podocytes. Both p-S473 and panAKT were normalized to nuclear marker DRAQ5^TM. (G) In-cell Western and quantification reveals that the ratio of phosphorylated ERK (p-ERK) to total ERK is significantly lower in CD2AP−/− podocytes compared to WT podocytes. Both p-ERK and total ERK were normalized to nuclear marker DRAQ5^TM. The experiments were performed three times with three replicates in each experiment. The bars show the mean expression in arbitrary units (error bars STDEV). *p < 0.05, ***p < 0.001, Student’s t-test.