Fig. 5. Expression of p46 Shc DN cancels growth inhibition and apoptosis induced by ALB6 treatment. (A) The total cell lysates of each p46 Shc DN clone were analysed by immunoblotting using anti-HA antibody. (B) MKN-28 cells, MKN-28V cells and p46 Shc DN1 cells at subconfluent density were serum starved for 24 hours and then stimulated with 50 µg ml^–1 ALB6 or isotype-matched mouse IgG1 for 10 minutes. The total cell lysates were immunoprecipitated with anti-Shc antibody and the blots were analysed for PY20 (top). The same filters were then stripped and reprobed with anti-Shc antibody (bottom). (C) The indicated cells were serum starved for 24 hours, and subsequently cultured for 48 hours in the presence of 50 µg ml^–1 ALB6, and their proliferation was measured using a [³H]-thymidine incorporation assay. Data are represented as the means±s.e.m. from four replications and are expressed as a percentage of the values found for cells cultured in medium alone. *P<0.01 vs control. (D) After the indicated cells (2x10⁴ cells per well) were cultured on six-well plates for 24 hours, the medium was replaced with fresh serum-deprived medium containing 50 µg ml^–1 ALB6. Their cell viability after ALB6 treatment for the indicated period was estimated by the trypan-blue dye-exclusion method. Data are represented as the mean cell viability (%) ±s. e.m. (n=4). *P<0.01 vs control. (E) MKN-28 cells, MKN-28V cells and p46 Shc DN1 cells were incubated with 50 µg ml^–1 ALB6, stained with annexin-V and analysed with a FACScan. Broken lines show the staining of untreated cells, solid lines show the staining of MKN-28 cells and shaded bars show the staining of p46 Shc DN1 cells. (F) The indicated cells were serum starved for 24 hours and then incubated with 50 µg ml^–1 ALB6 for 5 minutes. The total cell lysates were analysed by immunoblotting with the indicated antibodies The indicated cells were incubated with 50 µg ml^–1 ALB6 for 48 hours. The adherent cells were harvested and subjected to immunoblots with anti-caspase-3 antibody. The arrow indicates procaspase-3. The arrowhead indicates cleaved caspase-3. Each figure shows one of four similar experiments.