Fig. 5.

ZDHHC18 and ZDHHC23 regulate BMI1 polyubiquitination. a Expression levels of BMI1 and H2AK119Ub detected by western blot analysis in neural progenitor cells (NPC1 and NPC2), proneural glioma stem cells (GSCs) (PN12, PN16, and PN19), and mesenchymal GSCs (MES23, MES27, and MES29). β-actin was used as a loading control. b mRNA levels of BMI1 were detected by RT-PCR analysis in the proneural GSCs (PN12, PN16, and PN19) and mesenchymal GSCs (MES23, MES27, and MES29). β-actin was used as a loading control. Data are presented as means ± SEM. c BMI1 protein expression in PN12 and MES23 GSCs after cycloheximide treatment (CHX, 50 μM) for the indicated times. d, e Immunoprecipitation followed by immunoblotting was performed for determining BMI1 polyubiquitination in PN12 and MES23 GCSs in the presence and absence of lactacystin treatment for 5 h (Lacta, 10 μM) (d) or transfected with the indicated plasmids in the presence of lactacystin treatment for 5 h (Lacta, 10 μM) (e). f Lysates from 293 T cells expressing the FLAG-ZDHHC18, HA-ZDHHC23, and Myc-RNF144A were subjected to immunoprecipitation, followed by immunoblotting with anti-FLAG, anti-HA, and anti-Myc antibodies. g, h Lysates from PN12 (g) or MES23 (h) GSCs transfected with the indicated plasmids were subjected to immunoprecipitation with anti-ZDHHC23 (g), anti-ZDHHC18 (h), or anti-RNF144A antibody (g and h), followed by immunoblotting with anti-BMI1, anti-RNF144A, anti-ZDHHC18, and anti-ZDHHC23 antibodies