(A) Mass spectrometry analysis of Flag-SIRT2-interactingproteins after transfection of Flag-SIRT2 into HeLa cells. Lysates were immunoprecipitated with anti-Flag-agarose beads and eluted using Flag peptide, resolved by SDS-PAGE gel, and subjected to mass spectrometry analysis.
(B) Immunoblots of Flag-SIRT2 interacting proteins.
(C–F) Reciprocal co-immunoprecipitation of endogenous SIRT2 and CDH1 (C,D) or CDC20 (E,F) from immortalized SIRT2+/+ and SIRT2−/− MEF cells. After synchronization using serum starvation for 72 hr, cells were released into normal media for 16 hr and harvested for co-immunoprecipitation. The lysates were immunoprecipitated with anti-SIRT2, -CDH1, or CDC20 antibodies, respectively, and IPed samples were analyzed by immunoblotting with anti-SIRT2, -CDH1, or –CDC20, respectively.
(G,H) In vivo deacetylation analysis of endogenous CDH1 (G) and –CDC20 (H) from liver of SIRT2 WT and KO mice 48 hours after partial hepatectomy.
(See also Figure S4)