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. 2014 May 13;33(13):1438–1453. doi: 10.15252/embj.201386907

Figure 2. Reversible acetylation of BubR1 by CBP and SIRT2.

Figure 2

  1. 293T cells were transfected with vector, GCN5-FLAG, PCAF-FLAG, p300-HA, or CBP-HA and Western blotted for BubR1, FLAG, HA, and tubulin.
  2. 293T cells transfected with vector or CBP-HA with or without SIRT2-FLAG and Western blotted for BubR1, FLAG, HA, and tubulin.
  3. 293T cells were co-transfected with Myc-BubR1 and CBP-HA. Extracts were immunoprecipitated with anti-HA antibodies and Western blotted for Myc and HA.
  4. 293T cells were co-transfected with Myc-BubR1 and SIRT2-FLAG. Extracts were immunoprecipitated with anti-FLAG antibodies and Western blotted for Myc and FLAG.
  5. SIRT2 was immunoprecipitated from 293T cells with anti-SIRT2 antibodies and Western blotted for BubR1 and SIRT2.
  6. 293T cells were transfected with vector or CBP-HA. BubR1 was immunoprecipitated with anti-BubR1 antibodies and Western blotted for Ac-lysine and BubR1.
  7. Sirtuin enzymes and acetylated BubR1 purified from 293T cells were incubated in vitro with NAD+. Reactions were Western blotted for Ac-lysine, Myc, and FLAG.
  8. 293T cells were transfected with Myc-BubR1, CBP-HA, and either vector, wild-type, or catalytically inactive mutations of SIRT2-FLAG and SIRT3-FLAG. Cells extracts were immunoprecipitated with anti-Myc antibodies, and Western blotted for Ac-lysine, Myc, and FLAG.