Figure 4.

Fractionation of endogenous and ectopically expressed G proteins and caveolin from TX-100 extracts of MDCK epithelial cells. Stably transfected MDCK cells that ectopically express α_o (panel A) were compared with G418-resistant control cells that had been stably transfected with the empty vector (panel B). Four milliliters of detergent extract, adjusted to 40% sucrose, was loaded at the bottom of a tube followed by a 7-ml linear gradient of 30–5% sucrose. After centrifugation, 0.8-ml fractions were collected from the top of the gradients, half of which was acetone-precipitated and analyzed by Western immunoblotting. Most of the cellular protein (assayed by Ponceau S staining of the blots, not shown) remains below the gradient in the five bottom fractions (numbered 10–14). Ectopically expressed α_o (A) consistently comigrates with endogenous α_i, β, and caveolin (cav) in detergent-resistant, low-density fractions centered around fraction 6 (A and B). α_i was detected by B087 antiserum (1:10,000 dilution), β by B600 antiserum (1:10,000), α_o by culture medium from Mab 2A-producing cells (1:500), and caveolin (cav) by the purified polyclonal antibodies (30 ng/ml). Not shown are results for α_s, which comigrates with the other G protein subunits from MDCK and MA104 cells.