5 bromo 4 chloro 3 indolyl phosphate and nitrotetrazolium blue chloride

Cells were harvested at various time points post-infection, washed twice with cold phosphate-buffered saline (PBS), and lysed in lysis buffer (150 mM Tris [pH 7.5] containing 150 mM NaCl, 1% Triton X-100, and complete protease inhibitor cocktail [Roche]) and loading buffer (0.08 M Tris [pH 6.8] containing 2.0% SDS,10% glycerol, 0.1 M DTT, and 0.2% bromphenol blue). The solutions were boiled and vortexed for 10 min and then centrifuged at 14000 × g for 10 min. Supernatant proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes using a semidry apparatus (Bio-Rad). The membranes were probed with primary antibodies (anti-EV-D68 VP1 polyclonal antibody [Genetex, GTX132312] and anti-β-actin monoclonal antibody [Sigma, A3853]) at 4°C overnight, followed by incubation with secondary antibodies (alkaline phosphatase-conjugated goat anti-rabbit IgG [Jackson ImmunoResearch Laboratories, code:115-005-045] and goat anti-mouse IgG [Jackson ImmunoResearch Laboratories, code: 115-055-062]) for 1 h at 25°C. The membranes were stained with 5-bromo-4-chloro-3-indolyl phosphate and nitrotetrazolium blue chloride (Sigma-Aldrich) and visualized for band quantification.

Cell or supernatant samples were harvested and boiled in loading buffer (0.08 M Tris, pH 6.8, with 2.0% SDS, 10% glycerol, 0.1 M dithiothreitol [DTT], and 0.2% bromophenol blue), followed by separation on sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using a semi-dry apparatus (Bio-Rad, USA). Membranes were incubated with primary antibodies (anti-EV-D68 VP1 polyclonal antibody [Genetex, GTX132312]; anti-β-actin monoclonal antibody [Sigma, A3853]). Standard alkaline phosphatase-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies (goat anti-rabbit IgG [Jackson ImmunoResearch Laboratories, code:115-005-045]; goat anti-mouse IgG [Jackson ImmunoResearch Laboratories, code: 115-055-062]) were then used. The membranes were stained with 5-bromo-4-chloro-3-indolyl phosphate and nitrotetrazolium blue chloride (Sigma-Aldrich) and visualized for band quantification.