Protease inhibitor cocktail

Whole-cell lysates were extracted using RIPA buffer (Applygen Technologies Inc., Beijing, China) supplemented with a protease inhibitor cocktail (GlpBio Technology, Shanghai, China). The protein concentration was measured using a BCA protein assay kit (Elabscience Biotechnology Co., Ltd., Wuhan, China). Cell lysates were performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands were electrophoretically transferred onto PVDF membranes. Subsequently, non-specific antigen binding was blocked using 5% skim milk. The membranes were incubated at 4°C overnight with primary antibodies (1:1,000-diluted FDX1; Proteintech Group, Inc., Wuhan, China). Membranes were incubated the next day with 1:5,000-diluted secondary antibodies at room temperature for 1 h (Proteintech Group). Enhanced chemiluminescence was used to visualize the image (Proteintech Group). Antibody against β-actin was used as an internal reference (Proteintech Group).

To detect the interaction between VPS72 and KAT5, the steps of Co-IP were carried out as follows: HuH7 cells were first lysed in RIPA buffer supplemented with protease inhibitor cocktail (GlpBio technology, Montclair, CA, USA) for 30 min and centrifuged at 13,000 g for 5 min at 4°C. The collected supernatants were incubated with corresponding antibodies overnight at 4°C. Next, the 40 μL of protein A + G Agarose was added for 2 h incubation. After washing with PBS and boiled for 5 min, the 40 μL of protein samples were collected for the detection of western blot.

Confluent primary hBMEC monolayer was grown in 12-well plates, then washed three times with serum-free 1640 medium before infection at a multiplicity of infection (MOI) of 1. After 1 h of virus adsorption at 37 °C and 5% CO₂, the cultures were washed twice with serum-free 1640 medium to remove unbound virus, then cells were cultured in 2% fetal bovine serum 1640 medium at 37 °C with 5% CO₂ for 24 h and 72 h. Finally, cells were washed three times with pre-chilled phosphate-buffered saline (PBS), and subjected to RNA extraction using either a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) or RIPA (Epizyme, Shanghai, China) buffer with a protease inhibitor cocktail (GlpBio, Montclair, CA, USA) for Western blot analysis.