Mouse monoclonal anti flag

Vero E6 cells were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Carlsbad, CA, USA) supplemented with 5% or 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) at 37 °C in a humidified 5% CO₂ incubator (all reagents obtained from Invitrogen, Carlsbad, CA, USA). Herpes simplex virus-1 (HSV-1) strain F, vesicular stomatitis virus (VSV) strain Indiana, and porcine epidemic diarrhea virus (PEDV) strain CV777 were reproduced in Vero cells for 48 h. Rabbit monoclonal anti-DR5 were from Abcam (Cambridge, MA, USA). Human anti-Rabbit antibody-cleaved caspase-8 (p43), human anti-Rabbit antibody-cleaved caspase-3, and mouse monoclonal anti-FLAG was from Cell Signaling Technology (Danvers, MA, USA). The mouse monoclonal anti-GAPDH, FITC-conjugated goat anti-Mouse antibody, TRITC-conjugated goat anti-mouse antibody was from Proteintech (Rosemount, IL, USA). Mouse monoclonal anti-PEDV-N was from Alpha Diagnostic International (San Antonio, TX, USA). The pharmacological inhibitors Z-LE(OMe)TD(OMe)-FMK and Rh2 were from MedChemExpress (Monmouth Junction, NJ, USA). Annexin V- FITC/PI, Dead Cell Apoptosis kit was from Solarbio (Beijing, China).

Cells were harvested in EBC buffer (50 mM Tris at pH 8.0, 120 mM NaCl, 0.5% NP-40) containing protease inhibitors (Roche). Cell lysates were lysed and quantified by Bradford assay to obtain equal amount of protein extracts, then loaded and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto a polyvinylidene difluoride (PDVF) membrane (Bio-Rad). Membrane blot were incubated with specific primary antibodies as follows: Rabbit polyclonal anti-KIF1Bβ was a gift from Dr. Susanne Schlisio. Mouse monoclonal anti-FLAG, rabbit monoclonal cleaved caspase-3, rabbit monoclonal PARP, rabbit polyclonal caspase-9 were purchased from Cell Signaling Technology. Mouse monoclonal β-actin and rabbit polyclonal GAPDH was purchased from Santa Cruz Biotechnology. Primary antibody signals were detected using either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology).