RalB lysine fatty acylation promotes RalB-GTP binding. (A) GTP binding of RalB WT and RalB 8KR determined using the Sec5-RBD after 30 min starvation in EBSS buffer. (B) Quantification of the GTP binding assay. Statistical significance was determined using a student’s unpaired t-test with Prism7 software. (C) Live cell imaging of HEK293T cells co-overexpressing GFP-RalB (WT and 8KR) and mCherry-Sec5RBD. (D) Quantification showing the percentage of the Sec5-RBD membrane localization in cells overexpressing RalB WT and RalB 8KR. Statistical significance determined by a one-way Anova using Prism7 software, *** p < 0.001, ns= not significant. (E) Immunofluorescence of CTRL and SIRT2 KD HEK293T cells overexpressing Flag-RalB WT and Flag-RalB 8KR and endogenous Sec5 (F) Quantification of the number of Sec5 puncta in cells overexpressing RalB. Statistical significance was determined by a one-way Anova using Prism7 software *** p < 0.001, ** p < 0.01, * p < 0.05, ns= not significant.