Ab108921

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).

High-binding 96-well ELISA plates (Nunc) were coated with 0.5 μg/well of purified SARS-CoV-2 N protein in carbonate/bicarbonate buffer 0.05 M pH 9.6 and allowed to bind over night at 4 °C. Then, endogenous human NPC1 and HSP90 were purified using Immobilized Recombinant Protein G Resin (Generon) and 4 μg of specific antibodies against NPC1 (Abcam, ab108921) or HSP90 (Enzo Life Sciences, ADI-SPA-835) respectively. This procedure was performed as described in Co-IP assays except the elution step, that in this case was done with glycine 200 mM pH 2.5. Serial dilutions of endogenous NPC1 and HSP90 were added to the plate and capture was allowed to proceed for 1 h at 37 °C. Subsequently, plates were washed with PBS-T (PBS 0.1% Tween 20) and the binding of NPC1 to SARS-CoV-2 N protein was detected with a rabbit anti-NPC1 antibody (1:2000), revealed with an anti-rabbit HRP (1:2000) using a colorimetric substrate (OPD) and finally, quantified by absorbance at 492 nm in the EnSight multimode plate reader of PerkinElmer.