Fig. 2

a, a? Western blot analysis showing the protein levels of myc-tagged ARF3^WT and all the identified mutants in transfected COS-1 cells, basally and after treatment with cycloheximide (CHX) (10 ?g/ml) for the indicated time points (a), and with MG132 (100 ?M), or bafilomycin A1 (200 nM) for six hours (a?). b Pull-down assay using GGA3-conjugated beads shows ARF3 activation in COS-1 cells transiently transfected with WT or mutant myc-tagged ARF3 expression constructs. Active and total ARF3 levels are monitored using anti-myc antibodies. GAPDH and beta-tubulin are used as loading controls. Pull-down assays of ARF3^WT transfected cells performed in the presence of an excess of GDP and ?GTP are used as negative and positive controls, respectively (b, left panel). Pulldown samples in b (left and right), are loaded on different blots and processed parallelly. Representative blots are shown and data are expressed as mean ± SEM of three independent experiments. Two-way ANOVA followed by Tukey?s post hoc test (a, WT vs. all mutants, ****p < 0.0001; a?, K127E vs. K127E + MG132 **p = 0.0052; K127E vs. K127E + Bafilomycin *p = 0.0195; L12V/D67V vs. L12V/D67V + MG132 *p = 0.0123; L12V/D67V vs. L12V/D67V + Bafilomycin *p = 0.0411), One-way ANOVA followed by Sidak?s post hoc test (b left panel, WT vs. WT + GTP *p = 0.0197), One-way ANOVA followed by Dunnett?s post hoc test (b, WT vs. K127E **p = 0.0088; WT vs. L12V/D67V **p = 0.0058; WT vs. D93N **p = 0.0035; WT vs. T32N **p = 0.0075; WT vs. Q71L ****p < 0.0001; WT vs. T31N ***p = 0.0006) are used to assess statistical significance. c Summary table of the data obtained relative to the stability and activity of the different ARF3 mutants. Source data are provided as a Source Data file.