Fig. 2

SPOP promotes non-degradative ubiquitination of BRAF. a Western blot of indicated proteins in WCL from Ishikawa cells treated with DMSO, MLN4924 (100 nM), or with MG132 (20 μM) for 8 h. b Western blot of WCL from 293 T cells transfected with FLAG-BRAF and increasing doses of Myc-tagged SPOP-WT or deletion mutants. c Western blot of indicated proteins in WCL from Tet-on-inducible Ishikawa cells treated with doxycycline (DOX). d Western blot of the indicated proteins in WCL from Ishikawa or SPEC-2 cells with SPOP knockout through CRISPR/Cas9 methods. e, f Western blot of indicated proteins in WCL from 293 T cells treated with 50 µg/ml cycloheximide (CHX) and harvested at different time points. Data were shown as mean ± SD (n = 3) f. The p-values were calculated using the Two-way ANOVA test. n.s., non-significant. g, i 293 T cells were transfected with the indicated plasmids. 24 h after transfection, the WCLs were prepared and the in vivo ubiquitination assays were performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody. h 293 T cells were infected with lentivirus expressing SPOP-specific shRNA or negative control. The stable cell lines were transfection with HA-Ub and the cell lysates were prepared and immunoprecipitation was performed. The polyubiquitinated forms of BRAF were detected by Western blot with anti-HA antibody