Anti gp64 antibody

Cells were washed twice with PBS and lysed with 50 μl protein lysis buffer (Beyotime, Shanghai, China) containing 2% PMSF (Beyotime). After 30 min on ice, lysates were treated with 20% loading buffer (Beyotime) for 10 min at 100°C. Proteins were resolved by 15% SDS-PAGE and analyzed by immunoblotting using anti-Flag antibody (1:2000, Sigma, Burghausen, Germany) and anti-GP64 antibody (1:2000, Abcam, Cambridge, USA). The horseradish peroxidase-conjugated goat anti-mouse IgG (1:20000, Beyotime) was used as the secondary antibody. Protein bands were visualized using the Supersignal Western Femto Maximum Sensitivity Substrate Western Blotting Kit (Thermo, Waltham, MA, USA) and a Chemiluminescence Imaging System (Clinx Science Instruments, Shanghai, China).

Western blotting was performed as previously described (11 (link)). Cells were washed twice with PBS and lysed with protein lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail (MCE, USA) and 2% Triton X-100. After 30 min on ice, lysates were treated with 20% loading buffer (Beyotime) for 10 min at 100°C. Proteins were resolved by SDS-PAGE and analyzed by immunoblotting using anti-V5 antibody (1:2,000; Abcam, Cambridge, USA), anti-beta-tubulin antibody (1:1,000; abm, Canada), anti-Flag antibody (1:2,000; Sigma, Burghausen, Germany), anti-gp64 antibody (1:2,000; Abcam, Cambridge, USA), and anti-NPC2 antibody (1:1,000).