Fig 4.

Amino acid substitutions at position 71 reduce Rem stability and activity. (A) Substitutions at amino acid 71 inhibit MMTV-SP activity. Two different amounts of N-terminally GFP-tagged rem expression constructs were transfected as indicated. The values determined for the vector control and 12.5 ng of the L71P mutant were not statistically different (P = 0.055). The other values were statistically different from the control vector values (P = <0.05). (B) Mutation of amino acid 71 in the MMTV-SP affects protein stability. Expression constructs (250 ng) and the reporter vector pHMRluc were transfected into 293 cells and incubated for 36 h before addition of 10 μM MG132 for 12 h. Transfected cell extracts (50 μg) were used for Western blotting with GFP-specific or actin-specific antibody (upper and lower panels, respectively). (C) Luciferase assays of MG132-treated cells transfected with L71 mutants. Cells transfected with 12.5 ng of rem expression constructs were incubated in the absence or presence of MG132, and then extracts were tested for luciferase activity. Values are reported as described for Fig. 2.