Trans blot turbo transfect pack

To confirm the expression of GFP and GFP-N proteins, an SDS-PAGE and a Western blot (WB) was done. For the SDS-PAGE, Mini-PROTEAN TGX gels were used (Bio-Rad 4561096), then, the gels were transferred to PVDF membranes using the Trans-Blot Turbo Transfect Pack (Bio-Rad 1704159) and the Trans-Blot Turbo system (Bio-Rad). Following this, the transferred membranes were blocked in 10% skimmed milk powder dissolved in TBS-0.1% Tween (TBS-T) (50 mM Tris-HCl (pH 8.3), 150 mM NaCl and 0.5% (v/v) Tween-20) buffer for 1 h at room temperature. Primary antibodies NPC1 (Abcam, ab108921), HSP90 (Enzo Life Sciences, ADI-SPA-835), EEA1 (BD Biosciences, 610457) and SARS spike protein (Novus, NB100–56578SS) were diluted in 5% skimmed milk powder dissolved in TBS-T at 1:1000 with the exception of GFP (B-2) (sc-9996) at 1:4000, and then incubated at 4 °C overnight. After three washes, blots were incubated with appropriate anti-horseradish peroxidase (HRP) secondary antibody diluted in 5% skimmed milk powder dissolved in TBS-T at 1:5000 for 1 h at room temperature. Blots then were developed using enhanced chemiluminescence reagent (Bio-Rad) and detected with ChemiDoc™ XRS Gel Imaging System using Image Lab™ software (Bio-Rad).