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Fig. 1. Proteasome inhibitors prevent HEF1 degradation and result in p115 HEF1 accumulation in A1-F and MG-63 cells. (A) MG-63 and A1-F cells were plated onto FN-coated tissue culture dishes in DMEM supplemented with 10% FBS and grown to confluence. Cell layers were treated with 10 µM lactacystin (LAC), 50 µM LLnL, 50 µM ALLM or carrier solvent DMSO (Cont) for 14 hours. (B) MG-63 cells on FN-coated dishes were serum-starved for 6 hours and then treated with TGF-ß1 (2.5 ng/ml) for 12 hours to induce HEF1 expression (0 hours). TGF-ß1-containing medium was removed and cell layers were washed twice with serum-free medium. Cells were treated with 20 µg/ml cycloheximide (CHX) in the presence or absence of 10 µM lactacystin for indicated times. Cell lysates were prepared and processed for immunoblotting with anti-HEF1 pAb. Nitrocellulose membranes were stripped and reprobed with anti-p130Cas mAb as a loading control.

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