FIGURE 5:

Myristoylation and palmitoylation of Src and Fyn mutants correlate with their membrane interactions. COS7 cells expressing the indicated Src-GFP or Fyn-GFP proteins were metabolically labeled with [³H]myristate or [³H]palmitate as described under Materials and Methods. The Src-GFP or Fyn-GFP proteins were immunoprecipitated using sheep anti-GFP (α-GFP) antibodies and protein A/GammaBind-sepharose and subjected to fluorography (upper panels in A and B) for 20 d ([³H]myristate) or 2 mo ([³H]palmitate). The lower panels in A and B depict Western blots (rabbit anti-GFP) of Src- or Fyn-GFP from the immunoprecipitate of each sample. Sample processing was as described under Materials and Methods. (A, B) Fluorography (top panels) and Western blotting (bottom panels) results of typical experiments. (C, D) Quantification (mean ± SEM, n = 3) of the incorporation of [³H]myristate (C) and [³H]palmitate (D) into the GFP-tagged proteins. Bands for myristate or palmitate incorporation and of the GFP-tagged proteins were quantified by densitometry and normalized for protein level by the respective Western blot of the same immunoprecipitate. For [³H]myristate, the normalized value obtained for Src-GFP was taken as 100%; for [³H]palmitate, the analogous value obtained for Fyn-GFP was set as 100%. Asterisks indicate significant differences between the pairs indicated by the brackets (*, p < 10⁻²; Student’s t test).