Co-immunoprecipitation (Co-IP) and GST Pull-Down assays

For exogenous Co-IP assay, HEK293T or GC cells were transfected with different expression vectors for 48 h. For endogenous IP assay, the cells were inoculated on 10 cm culture plates. The cells were lysed with IP lysis buffer containing 50 mM Tris-HCl (pH8.0), 150 mM NaCl, 10 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 10% glycerol, 1 mM EDTA and protease inhibitor and incubated with anti-Flag (Sigma-Aldrich, F1804, 1:400) or anti-Myc antibody (Origene, TA150121, 1:400) for exogenous Co-IP, and anti-USP13 or anti-cyclin D1 for endogenous IP, then followed by the addition of Protein A/G Magnetic Beads (MedChemExpress, HY-K0202) overnight at 4 °C on a vertical roller. The beads were washed five times with PBST buffer. Both lysates and immunoprecipitates were analyzed by immunoblotting.

In the GST pull-down assay, GST-USP13 was expressed in Escherichia coli BL21 (DE3) cells, which were induced by isopropyl-b-D-thiogalactoside (IPTG, MedChemExpress, HY-15921). Cyclin D1 expression vector was transfected into HEK293T cells. The GST tagged USP13 protein was purified by Glutathione Sepharose beads (Yeasen, 20562ES08) and then subjected to immunoprecipitation assay.