Fig 1: RAB27B binds to ZDHHC9 and regulates NRAS palmitoylation via ZDHHC9.(A) 293T cells were transfected with constructs to express tagged RAB27B, NRAS and ZDHHC9 as indicated. Cells were then subjected to IP followed by WB using the indicated antibodies. (B) TF-1 DKO cells stably expressing HA-ZDHHC9 were subjected to coIP with anti-HA antibodies followed by WB analysis using the indicated antibodies to examine its interaction with endogenous proteins. (C) Schematic illustration of the hypothesis depicting how RAB27B regulates NRAS signaling (blue arrows) and how to restore NRAS signaling disrupted due to RAB27B loss (red arrows). (D–F) RAB27B-depleted TF-1 DKO cells stably expressing HA-ZDHHC9 or Myc-GOLGA7 were subjected to examination of palmitoylation status and cell growth. (D) WB to examine the efficiency of RAB27B KD or ZDHHC9 and GOLGA7 overexpression in TF-1 Ctrl and DKO cells. (E) APE assay to examine palmitoylation of endogenous RAS proteins. OE, overexpression; HAM, hydroxylamine; Palm, palmitoylated. (F) Cells were cultured in triplicate in different concentrations of human GM-CSF and cell growth after 3 days in culture was determined by MTT absorbance. Data are represented as mean ± SD. **P < 0.01; ***P < 0.001 compared with the shRAB27B group, determined by 2-way ANOVA.