Fig 2: Statins increase Rac1-GTP and cytokine production in a GGPP- and Iqgap1-dependent fashion. a Left, western blots showing levels of GTP-bound and total Rac1 in lysates of RAW 264.7 macrophages incubated for 3 weeks with Atorvastatin (5 µM), Rosuvastatin (2.5 µM), and Simvastatin (1 µM). Np-Rap1A was used as a marker of GGTase-I-deficient cells and Actin as a loading control. Middle and right, amounts of GTP-bound and total Rac1 determined by densitometry. b Left, western blots showing levels of GTP-bound and total RhoA in the same cells as in (a). Middle and right, amounts of GTP-bound and total RhoA determined by densitometry. c Il-1ß concentration, before and 8 h after LPS stimulation, in medium of RAW 264.7 macrophages incubated with Atorvastatin (5 µM) and Rosuvastatin (2.5 µM) for 21 days. GGPP (10 µM) was added to the cells 3 days before LPS stimulation. d Similar experiment as in c performed with J774 macrophages. e Cytokine concentration in medium of LPS-stimulated Iqgap1+/+ and Iqgap1-/- macrophages incubated with Simvastatin (5 µM) for 60 h. 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

Fig 3: Editing the endogenous Rac1 CAAX-motif increases GTP loading and reduces total Rac1 levels. a Predicted amino acid sequence from sanger sequence results of PCR-amplified Rac1 DNA and cDNA fragments. The DNA/cDNA was from HEK-293 clones whose Rac1 gene had been edited at the CAAX sequence by CRISPR/Cas9-facilitated homologous recombination to prevent geranylgeranylation by GGTase-I. CM1–3, CAAX mutant clones; Ctr, un-edited control clone. b Left, western blot showing electrophoretic mobility of wild-type Rac1 in Rac1Ctr lysates, and nonprenylated (np) Rac1 in CM1–3 lysates. c Left, western blots showing amounts of GTP-bound Rac1, total Rac1, and the loading control Actin in lysates of the gene-edited cells. Middle and right, levels of GTP-bound and total Rac1 determined by densitometry; data are mean of three independent experiments for each cell line. d Left, western blots showing the distribution of Rac1 in cytosol, membrane, and nuclear fractions (designated C, M, and N, respectively) of the gene-edited cells. Rho-GDI1, integrin-a5, and histone H3 were used as markers for cytosol, membrane, and nuclear fractions, respectively. Right, levels of nuclear Rac1 determined by densitometry data of three independent experiments and expressed as percent of Rac1Ctr. e, f Western blots showing levels of total Rac1 and the loading control Actin in lysates of gene-edited cells isolated after incubation with 15 µM lactacystin for 12 h (e); and 50 µM MG-132 at different time points (f). g Left, immunoprecipitation (IP) of Rac1 in gene-edited cells followed by western blot for Ubiquitin. Direct western blots were performed on the same lysates (Input) to quantify total Rac1 levels. Actin was used as a loading control. Right, Bar graphs showing levels of Rac1-Ub3. h Immunoprecipitation (IP) of Rac1 in gene-edited cells followed by western blot for Iqgap1. Direct western blots were performed on the same lysates (Input) to quantify Iqgap1 and Rac1 levels. Actin was used as a loading control. Error bars represent s.e.m

Fig 4: Iqgap1 binds nonprenylated Rac1 and mediates GTP loading, cytokine production, and arthritis. a Left, immunoprecipitation (IP) of Rac1 in BM macrophage lysates followed by western blots for Iqgap1. Direct western blots were performed on the same lysates (Input) to quantify total Iqgap1 and Rac1 levels. Nonprenylated Rap1A was used as marker of GGTase-I-deficient cells; actin was used as a loading control. Middle and right, levels of Rac1-bound Iqgap1 was determined by densitometry of IP-western blots of BM macrophages from six mice/genotype. b Synovitis and erosion scores in joints of 12-week-old Pggt1b?/+ (n = 4), Pggt1b?/? (n = 9), and littermate Iqgap1–/–Pggt1b?/? (n = 7) mice. c Cytokine concentrations, 8 h after LPS stimulation, in medium of BM macrophages isolated from Pggt1b?/+, Pggt1b?/?, and Iqgap1–/–Pggt1b?/? mice (n = 3/genotype). d Left panel, Western blots showing levels of GTP-bound Rac1, total Rac1, and Iqgap1 in BM macrophages isolated from Pggt1b?/+, Pggt1b?/?, and Iqgap1-/-Pggt1b?/? mice. Right panels, levels of GTP-bound and total Rac1 determined by densitometry (n = 5/genotype). e Western blots of macrophage lysates isolated at base-line and 15 and 30 min after LPS stimulation. f Confocal microscopy images showing F-Actin staining of BM macrophages. Scale bars: 10 µm. Error bars represent s.e.m. The graph shows adhesive area of 20–30 macrophages in ten randomly selected fields analyzed in cells from two mice/genotype. Significance between groups were calculated with two tailed Student’s t test (a, c, d, f) and one-way ANOVA with Tukey’s post hoc test (b). n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001