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. 2021 Jul 15;12:4339. doi: 10.1038/s41467-021-24639-y

Fig. 5. PI(4,5)P2 binding is necessary for ARHGEF3 membrane targeting, cellular activity and function.

Fig. 5

a NIH3T3 cells were transfected for 24 h with EGFP-tagged wild-type or lipid binding-deficient mutant K342E/K348E of ARHGEF3. The transfected cells were then treated with 0.03% digitonin for 2.5 min to remove cytosolic proteins before fixation. Percentage of transfected cells with PM localization was quantified. Data are presented as mean ± SEM (n = 3). Scale bars: 5 µm. b HEK293 cells were transfected with wild-type or lipid binding-deficient mutants of ARHGEF3, serum-starved overnight, and then stimulated with 10% FBS for 30 min followed by cell lysis and western blotting. Western blot signals were quantified by densitometry to generate ratio of pAKT versus total AKT. Data are presented as mean ± SEM (n = 3 independent experiments). c HEK293 cells were transfected for 24 h with empty vector (control), wildtype, or K342E/K348E mutant of ARHGEF3. Cell lysates were subjected to active RhoA pulldown assay using GST-rhotekin beads, and analyzed by western blotting. Western blot signals were quantified by densitometry to generate the relative ratios of RhoA pulled down (active) and RhoA in lysates (total). Data are presented as mean ± SEM (n = 5 independent experiments). d Purified wild-type and K342E/K348E mutant ARHGEF3 were subjected to in vitro RhoA guanine nucleotide exchange assay. Three independent experiments were performed with similar outcome, and representative results are shown. e NIH3T3 cells were transfected for 24 h with wild-type or K342E/K348E mutant of ARHGEF3, followed by fixation and phalloidin/DAPI staining. Three independent experiments were performed with similar outcome, and representative images are shown. Scale bars: 5 µm. Two-tailed Student’s t test was performed to compare WT and mutant in (a), and each sample to the control in (b, c). *P < 0.05; **P < 0.01; ***P < 0.001. P values: a 0.0001. b 0.0164, 0.0039, 0.0008, 0.0000, 0.0039. c 0.020, 0.038. Source data for (a–d) are provided as a Source Data file.