Figure 2

Src and PKC are required for αvβ3-dependent invasion. A) C8161.9, M14, or normal cultured melanocytes (NHEM) were placed in suspension (S) or plated on vitronectin (VN). The level of Src activity present in Src immunoprecipitates was measured by immunoblotting with rabbit anti-phospho-specific antibodies to the activation loop phosphorylated tyrosine, Y418 (Y418P Blot). Total levels of Src in the same immunoprecipitates were measured by immunoblotting stripped blots with total mouse anti-Src antibodies (Src Blot). The mouse immunoglobulin band in the immunoprecipitates is indicated (IgG). B) C8161.9 cells expressing the avian retroviral receptor tVA were left uninfected (C), or infected with avian retroviruses expressing GFP (G), wild type Src (WT) or a dominant interfering Src mutant (K-M or K295M). Levels of Src expression were monitored by immunoblotting of whole cell lystates (Src). Vitronectin-induced Matrigel invasion of Src-inhibited cells was quantified. Data represents 6 independent experiments. C8161.9 cells were also treated with two Src inhibitors, PD173955 (PD-R) or PD180970 (PD-S) and the extent of VN-induced invasion in the absence of serum or growth factors was assessed. C) Three melanoma cells lines, C8161.9, A375, M14, and normal cultured melanocytes (NHEM) were untreated (-) or pretreated (+) with 100 nM TPA for 16 hours to down regulate expression of classical and novel PKCs. The levels of PKCα, PKCδ, and PKCε expression were measured by immunoblotting of whole cell extracts. Equal levels of cell extract were loaded and probed by immunoblotting with specific antibodies. D) C8161.9 cells were pretreated with DMSO, TPA for 16 hours, or bisindolylmaleimide and vitronectin-induced Matrigel invasion in the absence of serum or growth factors was assessed and quantified.