Fig. 1
VMP1 is palmitoylated at cysteine 263 and 278 by ZDHHC3. A Human Sertoli cells (SCs) were metabolically labeled with 17-ODYA and then reacted with biotin-azide using click chemistry. Immunoprecipitation of biotinylated (palmitoylated) proteins from SCs showed VMP1 was palmitoylated and palmitoylation was suppressed by palmitoylation inhibitor 2-bromopalmitate (2-BP, 50 μM). Right graph: Quantified data of ratios of palmitoylated VMP1 to total VMP1 protein levels in cells treated with 2BP or control medium. ***P < 0.001. B Palmitoylation assay of VMP1 in SCs, HEK293T, PC3, LNCap cells that were either treated with DMSO (control) or 100 μM 17-ODYA. C Schematic diagram of VMP1 showed its potential palmitoylation sites and special domain (VTT domain). D Sequence alignments of VMP1 potential cysteine residues across different species. E SCs transfected with flag-tagged VMP1 or empty vector was tested with palmitoylation assay. F SCs transfected with flag-tagged VMP1 was tested with palmitoylation assay when NH2OH or Tris were added to prove palmitoylation occurred exclusively on cysteine residues. G VMP1 and cysteine mutant palmitoylation assays. SCs transfected with wild type flag-tagged VMP1 and the indicated single cysteine mutants were assayed for VMP1 palmitoylation levels with click chemistry. *P < 0.05. H SCs transfected with wild-type flag-tagged VMP1 or double cysteine site mutants were assayed for VMP1 palmitoylation level with click chemistry. ***P < 0.001. I Real-Time qPCR detected the mRNA of zDHHC2, 3, 4, 6, 7, 8, 11, 12, 15, 16, 20, 21, 24, gene expressions were normalized to GAPDH. *P < 0.05. J Venn diagram showing the expression patterns and subcellular distribution of different ZDHHCs in SCs. K SCs treated with indicated siRNAs targeting potential ZDHHCs (ZDHHC2, 3, 6, 7, 11, 15, 20, 21) for 72 h were assayed for VMP1 palmitoylation levels. L VMP1 palmitoylation was detected in HEK293T and SCs transfected with His-tagged ZDHHC3 vector or control vector. Lysate GAPDH served as a loading control