Mouse anti his

The protein concentrations were calculated using the Bradford assay (Bio-Rad). Whole cell lysates of BHK21 cells were collected at 24 h or 48 h post-transfection by three rounds of freeze-thaw. Unless otherwise stated, a total of 20 µg of the total cellular protein was boiled in 6× protein loading buffer before separation by 12% SDS-PAGE electrophoresis. Then, the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in TBST overnight at 4 °C, and subsequently incubated for 1 h with mouse anti-His (Proteintech, Shenzhen, China), Rabbit anti-actin monoclonal antibodies (Bioss, Beijing, China) or mouse anti-GAPDH monoclonal (Ruiying Biological, Suzhou, China) antibodies at a 1:2000 dilution. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG (Earthox, San Francisco, CA, USA) was used as the secondary antibody at 1:5000 dilution. Proteins were visualized by chemiluminescence using an ECL kit (Bio-Rad).

Rabbit anti-Rab5C (Proteintech, Cat. No. 27219-1-AP) and rabbit anti-TBC1D16 (Abcam, Cat. No. ab104407) were used at a dilution of 1:1000 for Western blotting. The anti-β-actin (Abcam, Cat. No. ab3280) were purchased from Abcam and used at a dilution of 1:10000 for Western blotting. Mouse anti-His (Proteintech, Cat. No. 66005-1-lg), mouse anti-Myc (Cell Signaling Technology, Cat. No. 2276S), rabbit anti-FLAG (Cell Signaling Technology, Cat. No. 14793S), mouse anti-FLAG (Cell Signaling Technology, Cat. No. 8146S) and rabbit anti-HA (Cell Signaling Technology, Cat. No. 3724S) were used at a dilution of 1:50 for immunoprecipitation. Antibody against PFV Gag was generously provided by Professor Li Zhi. The anti-Tas was produced by immunizing mice with prokaryotically expressed Tas, followed by purification according to standard procedures (18 (link)). HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit secondary antibodies were from Bioprimacy and used at 1:10,000.