Extended Data Fig. 7: Characterization of the bound-lipid in the DHHC9-GCP16 complex.
From: Regulation of RAS palmitoyltransferases by accessory proteins and palmitoylation
a, Brief scheme summarizing the experimental procedure for lipid extraction (see also Methods). b, Extracted ion chromatography (EIC) of PA (36:3e), PA (38:4e) and PA (36:4e). c, Data-dependent tandem mass spectrum of PA (36:3e), PA (38:4e) and PA (36:4e). Abbreviation: NL, neutral loss. d, Dot and bar plot showing the normalized intensities (in 71.5 µg protein samples) of detected phosphatidic acid (PA) species from three extraction and mass spectrometry experiments. e,f, Evaluation of the role of different phospholipids on the catalytic activity of the DHHC9-GCP16 complex. The DHHC9-GCP16 complex was immobilized on anti-FLAG beads and subsequently washed with a buffer containing 0.1% Triton X100 in order to remove the bound lipids. The complex was then eluted from the beads by FLAG peptide and incubated with various phosphatidic acids (PA), phosphatidylcholines (PC), or a buffer control. The catalytic activity was evaluated using the NBD assay. The data in (e) are the results of a representative experiment from three independent experiments. The data in (d) and (f) represent the mean ± SD of three independent measurements. The data in (f) were analyzed using the unpaired t test in Prism to calculate the two-tailed P values: **, P < 0.01; ns, P ≥ 0.05.
