Western blot of Syntaxin-4 was performed by loading 60 µg of WT (lane 1) and STX4 CRISPR KO (lane 2) HAP1 cell lysates in RIPA buffer onto a 4-15% gradient polyacrylamide gel. Proteins on the blots were visualized with Ponceau staining (below immunoblot). Proteins were transferred to nitrocellulose membrane and blocked in 5% milk for 1 hr. STX4 was detected at approximately 34 kDa (designated by the black arrow) using a STX4 recombinant monoclonal antibody (Product # MA5-38349) at a dilution of 1:1,000 in 5% BSA in TBS with 0.1% Tween 20 (TBST) overnight at 4°C. The peroxidase-conjugated secondary antibody (Product # 65-6120) was diluted to 0.2 µg/mL in TBST with 5% milk for 1 hr. Chemiluminescent detection was performed using Pierce ECL Western Blotting Substrate (Product # 32106). Data courtesy of YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies using knockout validation.