Effect of Nck2 on phosphotyrosine proteins in human melanoma cells. (A) Tyrosine phosporylated proteins in total cell lysates (30 μg protein) prepared from human primary (WM278) or metastatic (WM1617, WM164) melanoma cells, as well as from WM278 cells overexpressing GFP (C2) or GFP-Nck2 (N7, N14), treated with or without pervanadate (Na3VO4 at 100 μM, 15 min at 37°C), were evaluated by western blot using specific p-Tyr antibody. β-tubulin was probed as loading control. (B) Tyrosine phosporylated proteins coimmunoprecipitated with Nck2, pan-Nck or normal rabbit IgGs from human primary WM278 and metastatic WM1617 melanoma cell lysates were revealed by western blot using specific p-Tyr antibody. Arrow heads show proteins specific to or increased in Nck2 and pan-Nck IPs. Results shown are typical of 3-5 independent experiments.