P10013

For whole cell lysates, HAECs (2–3 × 10⁶ cells) were lysed with 100 μL lysis buffer (P10013, Beyotime) supplemented with 2% protease inhibitors and 2% phosphatase inhibitors (P1050, Beyotime) for 10 min on ice. The lysates were centrifuged at 13,000× g for 10 min at 4 °C. Protein concentration of the collected supernatants was measured by BCA Protein Assay Kit (23225, Thermo Fisher Scientific). Nuclear and cytoplasmic lysates were prepared by Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime). Equal amounts of protein samples (10 μg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane, blocked with 5% nonfat milk in TBST buffer at room temperature for 1–2 h, and analyzed by immunoblotting. Membranes were incubated with specific primary antibodies (for details, see Table S1) overnight at 4 °C and washed three times in TBST buffer before incubation in HRP-conjugated secondary antibody (111-035-003/115-035-003, Jackson Immunoresearch, West Grove, PA, USA). Membranes were washed three times again in TBST buffer before visualization with ECL substrate (1705061, Bio-Rad, Hercules, CA, USA). Images were acquired with the Clinx Chemi Analysis system (Clinx, Shanghai, China) and quantified with Image J software (National Institutes of Health, RRID: SCR_003070).

Cells were lysed in IP lysis buffer (P10013, Beyotime) containing protease inhibitor cocktail tablets for 15 min at 4 °C. After the protein concentrations were measured, equal levels of lysates were used for immunoprecipitation. Cell lysates were incubated with antibodies against MVP (sc-23916, Santa Cruz) or Fas (sc-74540, Santa Cruz) and protein A/G beads (sc-2003, Santa Cruz) overnight at 4 °C. Then, the precipitants were washed three times with IP lysis buffer, and the immune complexes were eluted with 2x loading buffer for 5 min at 95 °C, and subjected to western blot analysis. For ubiquitination assay, 4 h before cell harvest, 10 µΜ MG-132 (MCE, New Jersey, USA) was added into the medium.