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. 2010 Jan 19;30(7):1673–1688. doi: 10.1128/MCB.00540-09

FIG. 4.

FIG. 4.

BAF250b is ubiquitinated and degraded by the proteasome. (A) BAF250bCTD BC* mutants are degraded during immunoprecipitation. T7 antibody immunoprecipitates of transiently transfected T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA and BC*), with Myc-Cul2, were washed in a buffer containing 0.5 M KCl and 0.1% NP-40 and analyzed by immunoblotting with an antibody to T7 or Myc. The film exposure for the input panels was shorter than that for the IP panels. (B) MG132 and NEM stabilize BAF250b mutant proteins in the presence of Myc-Cul2. HeLa cells were transiently transfected with T7-BAF250b, T7-BAF250b with ARID deleted (ΔA), the T7-BAF250b BC box mutant (BC*), or T7-BAF250b with ARID deleted and the BC box mutated (ΔA, BC*) and with Myc-Cul2, treated with 5 μM MG132, and harvested in the presence of NEM. BAF250b proteins were immunoprecipitated with an antibody to T7 and probed for Myc-Cul2. (C) BAF250bCTDBC* is autoubiquitinated. T7 antibody immunoprecipitates of cells transiently transfected with vector (−) (lanes 1, 5, and 9), T7-BAF250bCTDWT (+) (lanes 2, 6, and 10), T7-BAF250bCTDBC* (lanes 3, 7, and 11), or T7-BAF250bCTDBC** (lanes 4, 8, and 12) and Myc-Cul2 (lanes 1 to 12) were washed in a buffer containing 0.3 M KCl and 0.5% NP-40 and immunoblotted for ubiquitin (endogenous), Myc, and T7.