Fig. 2.

Loss of SAFB reduces levels of FNTA and mislocalizes prenylated RAS and RHO family GTPases. The genomes of U2OS cells were edited by CRISPR-Cas9 and either nontargeting guides (−) or guides targeting SAFB (+) and stably transfected with an empty vector or plasmids directing expression of SAFB or FNTA. (A) Cell lysates were analyzed for the indicated prenyltransferase subunits by immunoblots. Data shown are representative of six independent experiments. (B) Cells were transiently transfected with the indicated GFP- or mCherry-tagged small GTPases and imaged alive by confocal microscope with or without FTI treatment (25 µM L-744,832). (Scale bar, 20 µm.) (C) Membrane association of endogenous RAS and RAP1 determined by subcellular fractionation with or without silencing SAFB as in A with or without overexpression of FNTA as indicated. Cells were disrupted by nitrogen cavitation, the postnuclear supernatant was separated into cytosol (S = supernatant) and total membranes (P = pellet) and the indicated proteins assayed by immunoblot. The percentage of RAS and RAP recovered in S vs. P for each condition is shown above each lane as determined by Li-Cor Odyssey scan. Data shown are representative of two independent experiments.