Figure 2.

RalB contains Lysine Fatty Acylation. (A) Alk14 labeling of RalA and RalB with and without hydroxylamine treatment in HEK 293T Cells. The image shown is representative of three additional independent experiments to the data presented in Figure 1. (B) Quantification of the relative level of hydroxylamine-resistant fatty acylation of RalA and RalB shown in A for three additional independent experiments. Quantification was done as described in Figure 1. (C) Alk14 labeling of RalB WT and the 8KR mutant in HEK293T cells. The data shown is representative of two independent experiments. (D) Quantification of fluorescence labeling of RalB WT and the 8KR mutant shown in (C). For quantification, the total florescence signal for the 8KR mutant was normalized to its protein level as determined by a flag western blot or Coomassie blue staining and was then divided by the normalized florescence signal for WT RalB. The normalized (fluorescence signal/protein loading) for RalB WT was set to 1. (E) Alk14 labeling before and after hydroxylamine treatment of RalB WT, the non-prenylated (RalB SC) and non-S-palmitoylated (RalB CS) RalB mutants in HEK293T cells. The image shown is representative of three independent experiments. (F) Fatty acylation of endogenous RalB in HEK293T cells. (G) Tandem MS/MS spectra of a doubly charged Alk14 modified peptide on RalB. (H) C-terminal sequences of RalB lysine to arginine mutants generated. (I) Relative hydroxylamine-resistant fatty acylation levels of the various RalB lysine to arginine mutants in HEK293T cells. Statistical significance was calculated using Prism 7 software using an ordinary one-way ANOVA. ** p < 0.01, *** p < 0.001. (p=0.0008 for the difference between RalB-WT and RalB-8KR, and p=0.0034 for the difference between RalB-WT and the RalB-M1/M5 mutant.) Fl, fluorescence; WB, western blot.