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Fig. 5. PMA inhibits the IL-3-induced tyrosine phosphorylation of FAK and FAK-binding proteins. Cells were deprived of IL-3 for 6 hours or grown on IL-3 and treated with 4 µM cytochalasin D (CytD), 1µM GF109203X (GF), 100 nM PMA alone or in combination for 30 minutes. (A) After immunoprecipitation with monoclonal anti-FAK antibody (IP:FAK) or non-immune mouse IgG1 (IP:IgG1) in denaturing conditions, lysates were analyzed on western blots with a mixture of anti-phosphotyrosine 4G10 and PY20 antibody (IB:PY), a phosphospecific anti-FAK(pY397) antibody (IB:FAKpY397) or polyclonal anti-FAK antibody (IB:FAK). (B) In another series of experiments, after immunoprecipitation with anti-FAK antibody (IP:FAK) or non-immune mouse IgG1 (IP:IgG1) in non-denaturing conditions lysates were analysed on western blots with phosphotyrosine PY20 antibody (IB:PY), anti-paxillin antibody (IB:paxillin) or anti-FAK antibody (IB:FAK).