Figure 6.

βγ complexes are enriched in detergent-resistant membrane domains. (A) IMR-32 cells (5 × 10⁶) were lysed directly in PBS/1% SDS/2 mM DTT; alternatively an equal amount of cells was first extracted with TX-100–containing lysis buffer, followed by re-extraction of the detergent-resistant membrane fraction in PBS/1% SDS/2 mM DTT. Total cell lysate and TX-100–resistant pellet were heated twice for 3 min at 100°C in the presence of SDS, and passed through a 21-gauge needle several times. Laemmli sample buffer was added and samples were analyzed after SDSPAGE and immunoblot for the presence of G protein β subunits. (B) Equal numbers of cells were cultured for 16–18 h in the presence (+) or absence (−) of 20% FCS. Total cell lysates and detergent-resistant membrane pellets were generated as described in A. Samples were normalized for the amount of protein (100 μg/lane) using the BCA protein assay. Samples were analyzed after SDS-PAGE and immunoblot, using the anti-β mAb ARC5.