Figure 2
Inhibition of SHIP2 activity reduces ROS production in CD2AP knockout podocytes but does not protect from apoptosis. (A) Representative immunoblot for SHIP2 in wild type (WT) and CD2AP−/− podocytes. Tubulin is included as a loading control. (B) Quantification of SHIP2 expression level in WT and CD2AP−/− podocytes in three replicate blots as in (A) shows an increase in SHIP2 expression. Tubulin was used for normalisation. (C) SHIP2 activity is increased in the absence of CD2AP. Treatment of CD2AP−/− podocytes with SHIP2 inhibitor AS1949490 reduces SHIP2 activity. (D) Representative immunoblot for SHIP2 of immunoprecipitations carried out with SHIP2 IgG (IP SHIP2) or goat IgG (IP IgG) as a control from 500 µg of protein lysates prepared from WT podocytes and CD2AP−/− podocytes treated or not with AS1949490. Similar immunoprecipitations were performed to enrich SHIP2 for activity assays. Full width blot is shown in Supplemental Fig. S1B. (E) Inhibition of SHIP2 prevents the increase in ROS generation induced by the absence of CD2AP, as observed by DCFH-DA fluorescent probe assay. Hoechst 33342 was used for normalization. (F) Flow cytometry with annexin V labelling indicates that AS1949490 treatment of CD2AP−/− podocytes induces apoptosis. (G) Representative immunoblot for PDK1 and CDK2 in WT and CD2AP−/− podocytes. Tubulin is included as a loading control. (H) Quantification of PDK1 and CDK2 in three replicate blots as in (G) in WT and CD2AP−/− podocytes shows a decrease in PDK1 and CDK2 expression in the absence of CD2AP. (I) SHIP2 inhibitor AS1949490 treatment reduces the expression of PDK1 in CD2AP−/− podocytes but not in WT podocytes. The experiments were performed three times with four (A–D,F–I) or 32 (E) 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.
