Horseradish peroxidase hrp conjugated anti flag antibody

NPC1 binding ELISAs were performed as described previously (4 (link)). Plates were coated with GP-specific monoclonal antibody (Mab) KZ52 (14 (link)) diluted to 2 μg/ml in phosphate-buffered saline (PBS). VSV-GP(WT) or VSV-GP(R64A) was normalized for GP content and then incubated at 37°C for 4 h in the presence of 500 μg/ml THL (Sigma-Aldrich, St. Louis, MO). The reaction was stopped by addition of 10 mM phosphoramidon. ELISA plates were blocked using PBS supplemented with 3% bovine serum albumin (PBSA; Thermo Fisher Scientific, Waltham, MA). THL-treated virus was added to blocked KZ52-coated plates and allowed to adsorb at 37°C for 1 h. After the plates were washed with 3% PBSA, a dilution series of FLAG-tagged NPC1-domain C was added and allowed to bind for 1 h at 37°C. Bound domain C was then detected using a horseradish peroxidase (HRP)-conjugated anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) and the Ultra-TMB substrate (Thermo Fisher Scientific, Waltham, MA). Half-maximal effective concentration (EC₅₀) values were calculated from two independent experiments, each with three technical replicates.

Dulbecco’s Modified Eagle Medium was from Cellgro. Fetal bovine serum was from Sigma-Aldrich. Restriction enzymes were from New England Biolabs. Protease inhibitor cocktail was from EMD Millipore Corporation (Cat#: 539131) and phosphatase inhibitor cocktail was from Cell Signaling Technology (Cat#: 5870). Horseradish peroxidase (HRP)-conjugated anti-Flag antibody was from Sigma-Aldrich (Cat#: A8592). Rabbit anti-MYPT1/2 was from Abcam (Cat#: ab32519). Anti-PPP1CB and anti-Phospho-MLC-2v (Ser15) antibodies were from Thermo Fisher Scientific (Cat#: PA5-28225 and PA5-104265, respectively). Anti-PPP1CC antibody was from Origene (Cat#: TA308937). Anti-Phospho-MYPT1 (Thr696) antibody was from Cell Signaling Technology (Cat#: 5163). Anti-MYBPC3 (Cat#: sc-137237) and HRP-conjugated anti-β-actin antibody (Cat#: sc-47778-HRP) were from Santa Cruz Biotechnology. Peroxidase-conjugated Goat anti-Rabbit IgG was from Jackson ImmunoResearch (Cat#: 111-035-003). SuperSignal West Pico Plus was from Pierce (Cat#: 34580). IRDye 680RD Goat anti-mouse and IR DYE 800CW Goat anti-Rabbit and Intercept Blocking Buffer were from LI-COR. Other general chemicals and supplies were from Thermo Fisher Scientific or Sigma-Aldrich.