Lipofectamine

AsPC-1 cells (5 × 10⁶) were transfected with plasmids encoding pcDNA3-Myc-XIAP and HA-Ub using Lipofectamine® 2000 for 48 h. Cells were pre-incubated with 2.5 μM MG132 for 1 h and then treated with BNTX (10 μM) and TRAIL (25 ng/ml) for 3 h. They were then lysed with an IP buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% NP-40, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, and 1 µg/ml leupeptin] containing complete protease and phosphatase inhibitor cocktails (Roche, Basel, Switzerland) for 0.5 h. After centrifugation, cell lysates were incubated with anti-Myc antibody (1 μg) overnight at 4°C on a rotator and then incubated with Protein G agarose beads (100 μl) for 2 h. Following a second centrifugation, the cell pellet was washed three times with the lysis buffer and boiled with a 2 × Laemmli sample buffer for 5 min. The ubiquitination of Myc-XIAP protein was detected by western blotting using anti-HA antibody.

BmN cells (1*10⁵) were transfected with 2 μg pIZT-V5/his vector and pIZT-V5his-ie-1mut with Lipofectamine (Roche, Mannheim, Germany), respectively. 48 h post-transfection, the cells were harvested and lysed in RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China). The expression levels of IE-1 and enhanced green fluorescence protein (EGFP) were detected with anti-His, anti-tubulin, and anti-EGFP antibodies (Proteintech, Rosemont, IL, USA) as the primary antibodies, and horseradish peroxidase-conjugated goat anti-mouse IgG (Proteintech, USA) was used as the secondary antibody. The reactions were visualized using an ECL reagent (Sangon, Shanghai).