Fig. 2

MUS81 regulates β-TRCP-mediated WEE1 ubiquitination. a and b PCR analysis of MUS81 and WEE1 after MUS81 knockdown in gastric cancer cell lines SGC7901 and BGC823. Data are presented as the mean ± SD (n = 3). c and d Western blot analysis of the impact of MUS81 knockdown on the expression of WEE1 at the protein level in gastric cancer cell lines SGC7901 and BGC823. e Representative images of WEE1 protein degradation after exposure of wild-type MUS81 (MUS81-WT) overexpression and parental AGS cells to cycloheximide (CHX; 50 μg/mL) for different time periods (top) and statistical line graph (bottom). Data are presented as the mean ± SD (n = 3). f Western blot analysis of the effect of chloroquine (10 μM) or MG132 (10 μM) on the degradation of WEE1 protein in MUS81-WT overexpression and parental AGS cells. G Ni-NTA pulldown analysis of 293 T cells transfected with the indicated plasmid to evaluate the effect of MUS81 on the ubiquitination of WEE1. His-Ub, His-tagged ubiquitin; oeMUS81-WT, FLAG-tagged wild-type MUS81; IB WEE1, immunoblotting with anti-WEE1 antibody. h Sketch map of MUS81 enzymatic mutant plasmid construction. Site-directed mutagenesis was used to replace two aspartic-acid residues within the MUS81 nuclease domain (338 and 339) with alanine residues. i Immunoblot of AGS cells transfected with the indicated plasmid, oeVector, FLAG-tagged wild-type MUS81 (oeMUS81-WT), and FLAG-tagged enzymatically inactivated MUS81 (oeMUS81-mut). j Co-immunoprecipitation was performed to detect the effect of MUS81 on the binding of E-3 ligase β-TRCP and WEE1