Figure 6

Reduction of TDAG51 expression in Ras ^V12 and Ras ^V12S35 -infected cells and their anchorage-independent growth. (A) Western blotting of TDAG51 protein (TDAG51) from 5 μg of cellular lysates from non-silencing vector control (pLVTHM) and anti-TDAG51 shRNA (36-1) -expressing cell lines as indicated. The blot was subsequently stripped and probed with an anti-alpha tubulin mAb (Alpha tubulin). Band intensities were determined using Image J software (NIH, Bethesda, MD) and TDAG51 values were normalized using alpha tubulin values. Fold expression of each band relative to HME16C pLRT pLVTHM: pLRT pLVTHM (1.0), 36-1 (0.4); V12 pLVTHM (2.3), 36-1 (1.0); S35 pLVTHM (1.5), 36-1 (0.7). Similar experiments to that shown were performed 2 additional times with similar results (B) Growth of pLVTHM vector control or TDAG51 shRNA-expressing Ras^V12S35-transduced cells either in the presence of 0.2% DMSO control (left) or 0.25 μM PD153035 (right) in low-attachment plates, assessed by the alamar blue growth assay. RFU, relative fluorescence units. (C) Growth of pLVTHM vector control or TDAG51 shRNA-expressing Ras^V12-transduced cells either in the presence of 0.2% DMSO control (left) or 0.25 μM PD153035 (right) in ultra-low-attachment plates, assessed by the alamar blue growth assay. RFU, relative fluorescence units.