Rotavirus Protein Expression Kinetics

Confluent MA104-GCaMP6s and MA104-GCaMP6s-IP₃R-TKO cells were inoculated with SA11-mRuby (MOI 10) and then harvested in RIPA buffer at 2, 4, 6, 8, and 10 hpi (21 (link)). The RIPA buffer composition was 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, and 1 cOmplete EDTA-free protease inhibitor tablet (Sigma-Aldrich). Samples were boiled for 10 min at 100°C in SDS-PAGE sample buffer and separated on Tris-glycine 4%–20% SDS-PAGE gels (BioRad) and blotted to nitrocellulose, which was blocked with 10% non-fat dry milk in PBS (BLOTTO). Primary antibodies were diluted in 0.5% BLOTTO and were rabbit anti-rotavirus [strain Alabama, (1:1,000)] (21 (link)), rabbit antisera to SA11 NSP4-aa120-146 (1:2,000) (25 (link)), and mouse anti-GAPDH (1:2,000). Alkaline phosphatase-conjugated secondary antibodies were used at a dilution of 1:2,000 in 0.5% BLOTTO. Membranes were incubated with primary antibodies overnight and incubated with secondary antibodies for approximately 2 h. After washing off secondary antibodies in 0.5% BLOTTO, membranes were developed using alkaline phosphatase detection solution (50 mM Tris, 3 mM MgCl₂, 0.1 mg/mL p-nitro blue tetrazolium chloride, and 0.05 mg/mL 5-bromo-4-chloro-3-indolyl phosphate).