FIG. 1.

Steady-state levels of WT and mutant Src family kinases. (A) COS-1 or SYF cells were transfected with the indicated c-Src constructs, and at 44 to 46 h posttransfection, cell lysates were analyzed by Western blotting, using anti-c-Src, anti-phospho-c-SrcY527, and antiactin antibodies. (B) Quantification of c-Src expression levels in COS-1 cells, as determined by Western blotting, normalized to c-SrcWT expression (set to 1). Combined data from three independent experiments are shown. Error bars represent standard deviations. (C) Subcellular distribution of c-SrcWT and mutants. COS-1 cells cotransfected with the indicated c-Src constructs and NMT were subjected to cell fractionation. Equal volumes of the soluble (S) and membrane pellet (P) fractions were analyzed by Western blotting, using anti-c-Src and anti-caveolin-1 antibodies. (D and E) Steady-state levels of Src family kinases after 2-hydroxymyristate treatment. COS-1 cells transfected with the indicated constructs were treated overnight with 2-hydroxymyristate or dimethyl sulfoxide (DMSO), and cell lysates were analyzed by SDS-PAGE and Western blotting with appropriate antibodies. (F) Quantification of the experiment in panel E. For each kinase, the steady-state level after 2-hydroxymyristate treatment is shown relative to the DMSO control level, which was set to 1. The averages for three independent experiments are shown. Error bars represent standard deviations.