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. 2010 Apr 7;38(14):4607–4619. doi: 10.1093/nar/gkq227

Figure 2.

Figure 2.

SHP interacts and colocalizes with SIRT1. (A and B) In vivo interaction of exogenous SHP with exogenous SIRT1, SIRT6 and SIRT7 (A). 293T cells were cotransfected with Flag–SIRT1 (panel A, left), Flag–SIRT6 or Flag–SIRT7 (panel A, right) and pEBG–SHP (GST–SHP) or with pEBG (GST) alone. The complex formation (GST purification) and the amount of Flag–SIRT1, Flag–SIRT6 or Flag–SIRT7 used for in vivo binding assay (cell lysate) were determined by western blot using indicated antibodies. In vivo interaction of endogenous SIRT1 with endogenous SHP (B). Co-immunoprecipitation assays were performed with cell extracts from HepG2 cells (panel B, left) and C57BL/6J mouse liver tissue extracts (panel B, right). Endogenous SIRT1 or endogenous SHP was immunoprecipitated with SHP or SIRT1 respectively, and were analyzed by western blot using indicated antibodies. (C) Colocalization of SHP with SIRT1. HeLa cells grown on coverslips in 12-well plates were transfected with expression vectors encoding GFP–SHP and Flag–SIRT1 (200 ng each). For the immunofluorescence of fixed cells, Flag–SIRT1 protein was immunostained with mouse monoclonal anti-Flag antibody and visualized with dye Alexa Fluor 488-conjugated anti-mouse antibody. The cell images were captured under 400× magnifications. Data is representative of atleast three independently performed experiments.