Fig. 4: USP46 deubiquitinates and stabilizes NRAS activation mutants.
From: Pharmacological targeting of casein kinase 1δ suppresses oncogenic NRAS-driven melanoma
a USP46 and NRAS protein levels in SK-MEL-103 and SK-MEL-30 cells expressing shScramble (shScr) or shUSP46 (#1 and #2) were measured by western blotting. b NRAS mRNA levels in cells (a) was determined by qRT-PCR. SK-MEL-103 (c) and SK-MEL-30 (d) cells expressing shScramble (shScr) or shUSP46 were treated with DMSO or MG132 (10 μM) for 10 h and western blotting was performed. e Cycloheximide pulse-chase assay was performed in cells (a); NRAS protein levels relative to β-actin was measured by Image J. f SK-MEL-103 cells were cotransfected with empty vector (EV), pLV5-NRAS Q61R (containing HA and S tag), FLAG-USP46 WT or FLAG-USP46 C44S mutant, then treated with MG132 for 10 h. NRAS Q61R was pull-downed by S-protein agaroses and polyubiquitylated NRAS Q61R was detected. SK-MEL-103 and SK-MEL-30 cells were cotransfected with empty vector, pIRES-NRAS Q61R (g), pIRES-NRAS Q61K (h) (containing FLAG and S tag) and other indicated plasmids. His-tagged ubiquitin was pulled-down by Ni-NTA beads and polyubiquitylated NRAS Q61R/K protein was examined. i SK-MEL-103 cells were transfected with empty vector or pLV5-NRAS Q61R (containing HA and S tag) and treated with MG132 (10 μM) for 10 h. NRAS Q61R was pull-downed by S-protein agaroses and incubated with purified GST, GST-USP46 WT or GST-USP46 C44S at 4 °C for 24 h. Polyubiquitylated NRAS Q61R was examined. j SK-MEL-103 cells expressing shScramble (shScr) or shUSP46 were transfected with empty vector or indicated plasmids followed by MG132 treatment for 10 h. NRAS Q61R was pull-downed by S-protein agarose and polyubiquitylated NRAS Q61R was examined. k Cells expressing empty vector, pLV5-NRAS Q61R, K104, K117, K128, K135 or K147 mutants (containing HA and S tag) were transfected with empty vector or FLAG-USP46 and treated with MG132 (10 μM) for 10 h. NRAS Q61R was pull-downed by S-protein agarose and polyubiquitylated NRAS Q61R was examined. Immunoblots are representative of three independent experiments (a, e). Data were presented as mean ± SD of three independent experiments (b). Data were analyzed by two-sided one-way ANOVA in (b, e). Source data are provided as a Source Data file.
