Effect of Nck2 on human primary melanoma cell proliferation. (A) Total cell lysates from stable WM278 human primary melanoma cells overexpressing GFP (C2) or increasing levels of GFP-Nck2 (N15 < N7 < N14) were subjected to western blot analysis using GFP or Nck2 specific antibodies. Cell proliferation of (B) WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7 and N14), as well as (C) parental primary (WM278) and metastatic (WM1617) melanoma cells was determined by quantification of Crystal Violet incorporation each day during 4 days in culture. Results are shown as the ratio of the mean of cell density over day 1 ± SD (n = 4). * p < 0.02, ** p < 0.006 and ***p ≤ 0.0001compared with C2 (B) or parental WM278 cells (C) using Student's t-test. (D) Nck1 and CrkII expression was evaluated using total cell lysates (30 μg protein) prepared from WM278 cells stably overexpressing GFP (C2) or GFP-Nck2 (N15, N7, and N14). β-tubulin was used as loading control.