Stabilization of MEIS2 by O-GlcNAcylation through the suppression of its ubiquitination. a, b WB and semi-quantitative analysis of MEIS2 protein levels in shCtrl and shOGT HEPM cells. shOGT is short for short hairpin OGT (OGT lentivirus). c qRT-PCR results of MEIS2 mRNA levels in shCtrl and shOGT HEPM cells. d HEPM cells received 8 h treatment with 10 μmol/L MG132 after 12 h treatment with DMSO, 30 μmol/L Thiamet G or 30 μmol/L OSMI-1. MEIS2 expression were observed by WB. e, f Half-life analysis of MEIS2 in HEPM cells. After an indicated period of treatment with 250 μmol/L CHX, MEIS2 levels in cells were observed by IB. Cells were treated with DMSO, 30 μmol/L OSMI-1 or 30 μmol/L Thiamet G. g Half-life analysis of HA-MEIS2 in HEK293 cells. After an indicated period of treatment with 250 μmol/L CHX, HA-MEIS2 levels in HEK293 cells were observed by IB. HA-MEIS2 (WT or S237A) plasmids transfected cells. h, i Cells received treatment with DMSO, 30 μmol/L Thiamet G or 30 μmol/L OSMI-1–12 h, endogenesis (h) or exogenous (i) MEIS2 ubiquitination was observed by anti-MEIS2 or anti-HA antibodies immunoprecipitated. j Cells were subjected to transfection with HA-WT or HA-S237A plasmids and treatment with 30 μmol/L Thiamet G 12 h before harvesting. MEIS2 ubiquitination levels were detected by IP assays. n = 3 biologically independent experiments. Data were indicated by mean ± SD. P-values were computed by one-way ANOVA followed by Tukey’s multiple comparisons test, *P < 0.05 and **P < 0.01
|
