FIG 1.

cPLA₂α specifically interacts with SIRT2. (A) Identified peptide (interacting fragment) aligned with SIRT2 bp 660 to 1926. (B) Interaction of full-length cPLA₂α and the identified peptide in HEK293 cells. cPLA₂α interacts with PLIP but not with Kid1 as positive and negative controls, respectively. Relative molecular weights in thousands are given to the left. (C and D) Reciprocal interaction of HA-cPLA₂α with EGFP-SIRT2 in HEK293 cell lysates. Specificity was confirmed by using species-matched control IgG (Ctrl IgG). Input shows levels of cPLA₂α and SIRT2 in total lysate. (E) Reciprocal interaction of endogenous cPLA₂α and SIRT2 in wild-type MEFs by immunoprecipitation using anti-cPLA₂α and anti-SIRT2 antibodies in two different reactions. Specificity was confirmed by using species-matched control IgG (Ctrl IgG) and cPLA₂α^−/− MEFs as negative controls. (F) Purified GST or GST-SIRT2 was incubated with purified His-cPLA₂α. GST was immunoprecipitated, and His-cPLA₂α bound to GST-SIRT2 was examined by Western blotting. Coomassie staining shows the expression of different proteins in each reaction product. (G) Generation of deletion mutant constructs of SIRT2 tagged with T7 antigen (upper panel) and interaction of cPLA₂α with T7-tagged deletion mutants of SIRT2 in HEK293 cells (lower panel). cPLA₂α interacts with SIRT2^136–352 and SIRT2^136–224 but not with SIRT2^136–194 or the KRAB-A domain of Kid1 (Kid1A) (negative control). (H) Generation of deletion mutant constructs of cPLA₂α tagged with EGFP (upper panel) and co-IP between SIRT2 and EGFP-tagged deletion mutants of cPLA₂α (lower panel). IB, immunoblotting; LC, light chain; CDS, coding sequence; FL, full-length; Ab, antibody.