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. 2010 Jul 1;24(13):1403–1417. doi: 10.1101/gad.1901210

Figure 5.

Figure 5.

SIRT1 directly deacetylates SREBP and controls SREBP protein stability. (A) HeLa cells were transfected with Flag-tagged SREBP-1a, and then were treated with (+) or without (−) 20 mM NAM for 20 h. Flag-SREBP-1a was purified by immunoprecipitation using anti-Flag antibodies, and was eluted with Flag peptide. The presence of acetylated SREBP-1a was detected by immunoblotting using an acetyl lysine-specific antibody. Flag-SREBP-1a served as the loading control. (B) HeLa cells were treated with proteasome inhibitors alone or with proteasome inhibitors and 25 mM NAM for 6 h. Endogenous SREBP-1a was immunoprecipitated by antibodies specific to SREBP-1a. The presence of acetylated SREBP-1a was detected by immunoblotting using an acetyl lysine-specific antibody. (C) HeLa cells were treated with proteasome inhibitors alone or with proteasome inhibitors and 25 mM NAM for 6 h. Endogenous SREBP-2 was immunoprecipitated. The presence of acetylated SREBP-2 was detected by immunoblotting using an acetyl lysine-specific antibody. (D) Overexpressed Flag-SREBP-1a proteins were purified from HeLa cells treated with NAM, and were incubated with GST fusion proteins as indicated in the in vitro deacetylation assay. The degree of SREBP-1a acetylation was detected by immunoblotting using an acetyl-lysine-specific antibody. Flag-SREBP-1a served as the loading control. (E) HeLa cells were transfected with Flag-tagged SREBP-1a, and then were treated with (+) or without (−) 20 mM NAM for 20 h. After the indicated time of cycloheximide (0.1 mM) treatment, whole-cell extracts were prepared, and Flag-SREBP-1a was detected by immunoblotting using anti-Flag antibodies. Equal amounts of proteins were loaded in each lane. (F) Overexpressed Flag-SREBP-1a or Flag-SREBP-1a K324R, K333R were treated with nicotinamide and cycloheximide as in E. (G) IMR-90 cells were placed in 1% lipid-depleted serum with or without NAM for 16 h. Nuclear extracts were prepared and detected by immunoblotting with an antibody to SREBP-1. (FL) Full-length SREBP-1 precursor; (M) mature, nuclear SREBP-1. (H) Endogenous SREBP-2 in IMR-90 cells was detected as in G.