Fig. 5

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-α5, 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