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. Author manuscript; available in PMC: 2012 Dec 20.
Published in final edited form as: Cell. 2008 Jul 25;134(2):329–340. doi: 10.1016/j.cell.2008.07.002

Figure 5. Interaction of SIRT1 with CLOCK.

Figure 5

(A) JEG3 cells were cotransfected with a series of expression vectors as described. Flag-tagged SIRT1 proteins were immunoprecipitated by FLAG antibody, and abundance of coimmunoprecipitated proteins was determined by western blotting using anti-Myc antibody (top right panel) and anti-SIRT1 antibody (bottom right panel). Left panels show the immunoblotting results of total cell lysates as an input.

(B and C) SIRT1 and CLOCK interaction in vivo. Endogenous CLOCK:BMAL1 and SIRT1 interactions in the mice liver (B) and in cultured MEFs after serum shock

(C) were determined by coimmunoprecipitation assays.

(D) The deacetylase-deficient (H363Y) mutant SIRT1 interacts with CLOCK as well as WT SIRT1.

(E) CLOCK (aa 450–570) is required for the interaction with SIRT1. In vitro-translated [35S]-labeled truncated CLOCK proteins were pulled down by GST-SIRT1.

(F) SIRT1 N terminus (aa 1–231) is required for CLOCK interaction. In vitro-translated [35S]-labeled full-length CLOCK proteins were pulled down by truncated GST-SIRT1.