Anti tubulin antibody

Total cell extracts were harvested in radioimmunoprecipitation (RIPA) lysis buffer (Cat. No: R0010-100, Solarbio, China) supplemented with protease inhibitors on ice. Protein quantification was determined with a PierceTM BCA protein assay kit (Cat. No: 23227, Thermo Fisher Scientific, Waltham, MA, USA). Protein detection was performed using SDS-PAGE, and western blots were carried out according to standard methods. Proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA). The membranes were then blocked overnight with 5% non-fat dried milk for 2 h and incubated with anti-BAIAP3 antibody (Cat. No: 24836-1-AP, Proteintech, USA) at 1:5000 dilution, and anti-Tubulin antibody (Cat. No: 11224-1-AP, Proteintech, USA) at 1:50,000 dilutions overnight at 4° C. After washing with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween20), the membranes were incubated for 4 h at room temperature with goat anti-rabbit second antibody at 1:20,000. Finally, the protein bands were detected by enhanced chemiluminescence (ECL) (Advansta, USA) with a BioSpectrum Gel Imaging System (UVP, USA). All experiments were performed at least three times.

Madin–Darby Bovine Kidney (MDBK) cells were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 medium (1:1) (Gibco, Grand Island, NY, USA) supplemented with 7% fetal bovine serum (Thermo Scientific, Waltham, MA, USA) at 37 °C in a humidified atmosphere containing 5% CO₂.
The NCP-BVDV-BJ175170 isolate strain, which was isolated from blood samples from cows with suspected BVDV infections, was maintained in our laboratory. The BVDV BJ175170 isolate strains were utilized for all experiments and are represented by “BVDV” in this article.
FTA (purity > 99%) was purchased from Selleck (Houston, TX, USA), solubilized in 100% DMSO, and kept frozen as 10 mM stocks. The compounds were stored in small aliquots to prevent multiple freeze-thaws and were stepwise diluted to reach the desired concentration in 0.5% DMSO for all treatments. Anti-NLRP3 antibody and anti-ASC antibody, anti-Caspase-1 antibody, anti-Tubulin antibody (ProteinTech Group, Rosemont, IL, USA), and anti-BVDV E2-protein mouse monoclonal antibody (mAb) (VMRD, Pullman, WA, USA) were used in the study.