Figure 3.

Gα₁₂ modulates the levels of p27^Kip1 and Skp2. (A) Lysates (50 µg) from pcDNA3- and Gα₁₂QL-NIH3T3 cells, serum starved for 24 hours and stimulated with 5% CS for the indicated lengths of time, were separated on a 10% SDS-PAGE and subjected to immunoblot analysis using antibodies specific to p27^Kip1. The blot was reprobed with GAPDH antibodies to monitor equal loading of proteins. (B) Lysates (50 µg) from pcDNA3-and Gα₁₂QL-NIH3T3 cells that were serum starved for 24 hours and stimulated with 5% CS for 0, 4, and 8 hours were subjected to immunoblot analysis using Skp2-specific antibodies. The blot was probed with antibodies to GAPDH to monitor equal loading of proteins. (C) Lysates (50 µg) were prepared from 2 independent pcDNA3- and 3 Gα₁₂QL-NIH3T3 cell clones that were serum starved for 24 hours and then stimulated with 5% CS for 0, 6, and 12 hours. Immunoblot analysis was carried out using Skp2-, p27^Kip1-, or GAPDH-specific antibodies. The experiment was repeated 3 times; results from a typical experiment are presented. (D) NIH3T3 cells that were infected with 600 MOI of Gα₁₂QL expressing adenovirus or empty pShuttle adenovirus were serum starved for 24 hours and then stimulated with 5% CS for the indicated time points (0, 6, and 12 hours). The blot prepared using the lysates (50 µg) from these cells were sequentially probed with antibodies specific to Skp2, p27^Kip1, and Gα₁₂.