Figure 3.

RGS4 incorporates [³H]palmitate in a hydroxylamine-sensitive linkage. (A) Sf9 cells expressing RGS4-myc were metabolically labeled with [³H]palmitate or with [³⁵S]methionine. The protein was immunoprecipitated from detergent extracts of membranes (M) or from cytosol (C) with a mAb against the myc epitope. Immunoprecipitates were resolved by SDS/PAGE, and the gels were treated with either 1 M Tris (pH 7) (Tris) or 1 M hydroxylamine (pH 7) (HA). The fluorograph was exposed to film for 3 days. (B) Sf9 cells expressing RGS4-myc or C2A RGS4-myc were labeled with [³H]palmitate and immunoprecipitated from detergent extracts of membranes. After resolution by SDS/PAGE, the RGS4 bands were excised from the gel and subjected to base hydrolysis. Base hydrolysates were extracted into organic solvent and chromatographed over a reversed-phase Ultrasphere C18 column. Fractions were collected and quantitated by scintillation counting. Elution positions of standards [myristate (C14:0), palmitate (C16:0), and stearate (C18:0)] are indicated with arrows.