FIG. 6.

Tom1L1-CHC affects SrcY527F membrane partitioning, SrcY527F-induced DNA synthesis, and focus formation. (A) Tom1L1-CHC reduces SrcY527F level in CEF. Shown are the levels of avian Src and caveolin from CEF purified from HEK 293 cells that were transfected with SrcY527F together or not with the indicated Tom1L1 constructs. The levels of expressed Src, Tom1L1, and tubulin expressed from the whole-cell lysate (WCL) are shown. wb, Western blotting; αcaveolin, anticaveolin; αTom1L1.2, anti-Tom1L1.2. (B) Tom1L1-CHC inhibits Src-driven DNA synthesis. NIH 3T3 cells stably transformed by SrcY527F (Src 527), seeded onto coverslips, and transfected or not with the indicated constructs were incubated in 0.5% serum for 30 h and further incubated in the presence of BrdU for 18 h. Cells were then fixed and processed for immunofluorescence. The percentage of transfected cells that incorporated BrdU was calculated as described in Materials and Methods. Results are expressed as the mean ± standard deviation of three independent experiments. (C) Tom1L1-CHC inhibits SrcY527F transforming activity. The statistical analysis of inhibition of SrcY527F-induced foci by Tom1L1-CHC is shown. NIH 3T3 cells were transfected or not with siRNA CHC as shown and then infected with control (mock) or indicated retroviruses. After 12 days of growth, foci were stained and scored as described in Materials and Methods. Focus formation (percentage of foci obtained relative to foci induced by SrcY527F) is represented as the mean ± standard deviation of three independent experiments.