Fig. 2

GGTase-I knockout increases Rac1-GTP loading, but reduces Rac1 total levels. a Left, western blots showing steady-state levels of GTP-bound and total Rac1 in BM macrophages. Nonprenylated RAP1A was used as marker of GGTase-I-deficient cells; actin was used as a loading control. Middle and right, amount of GTP-bound (n = 5/genotype) and total Rac1 (n = 17/genotype) in BM macrophages determined by densitometry of protein bands. b Quantitative polymerase chain reaction (QPCR) data showing levels of Rac1, Rhoa, and Cdc42 expression in cDNA of BM macrophages (n = 3 per genotype). c Left, western blots showing levels of Rac1 and Actin that remain in BM macrophages at various time points after incubation with cycloheximide (20 µg/ml) to stop protein synthesis. Equal amounts of total proteins were loaded. Right, densitometry of protein bands normalized to time-point 0 within each genotype. d Similar experiment as in (c) except twice the amount of total proteins from the Pggt1b^Δ/Δ lysates were loaded compared to Pggt1b^Δ/+ to obtain similar Rac1 levels at time-point 0. e Left, immunoprecipitation (IP) of Ubiquitin (Ub) followed by western blots for Rac1. Direct western blots were performed on the same lysates (input) to quantify total levels of Rac1 and Actin. The molecular weight of the main band was ~45 kDa which corresponds to Rac1 conjugated with three Ubs. Two independent experiments are shown. Right, amount of ubiquitin-bound Rac1 determined by densitometry of protein bands (n = 4/genotype). f Upper panel, western blots showing total Rac1 levels in lysates of BM macrophages incubated for 10 h with proteasome inhibitors MG-132 (15 µM) and lactacystin (15 µM). Lower panel, quantification of protein bands normalized to Actin and expressed as percent of control (Pggt1b^Δ/+). Similar results were obtained three times. Error bars represent s.e.m. Significance between groups were calculated with two tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001