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. Author manuscript; available in PMC: 2012 Oct 22.
Published in final edited form as: Biochem J. 2011 Jan 1;433(1):245–252. doi: 10.1042/BJ20101293

Figure 2. XBP1s is deacetylated by SIRT1.

Figure 2

(A and B) HEK293 cells were transfected with Flag-XBP1s and p300. Cells were incubated for 4 hours in the absence or presence of TSA (5μM), NAM (10 mM), or EX-527 (1 μM). Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. The extent of acetylation was defined by the ratio of acetylated vs. total XBP1s from duplicate experiments (Fig.2A, lower panel). (C) HEK293 cells were transfected with Flag-XBP1s and p300 together with a pSuper-shRNA vector targeting SIRT1 or its empty vector. Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. (D) HEK293 cells were transfected with Flag-XBP1s and p300 together with or without SIRT1 expressing construct. O/E, overexpression. Cell lysates were analyzed by immunoprecipitation and Western blotting. *, non-specific band. (E) The acetylated Flag-XBP1s was purified from 293T cells transfected with Flag-XBP1s and p300. The Flag-SIRT1 was purified from 293T cells separately transfected with Flag-SIRT1. Purified acetylated XBP1s was incubated in the presence or absence of SIRT1 and/or NAD+. The acetylated and total amounts of XBP1s were determined by Western blotting and the extent of acetylation was defined by the ratio of acetylated vs. total XBP1s from duplicate experiments.