Immobilized recombinant protein g resin

Co-IP for NPC1 was performed using 50 μl of the Immobilized Recombinant Protein G Resin (Generon) and specific antibodies against NPC1 (Abcam, ab108921) as previously described (García-Dorival et al., 2016 (link)). The cell pellets were incubated for 30 min on ice with 200 μl lysis buffer. The lysate was then clarified by centrifugation and diluted five-fold with dilution buffer prior to adding 2 μg of the primary antibody and then incubated at 4 °C on a rotator for 2 h. The protein G resin (Generon) were equilibrated with ice-cold dilution buffer and then incubated at 4 °C on a rotator with diluted cell lysate containing the antibody overnight at 4 °C on a rotator, followed by centrifugation at 2500×g for 2 min to remove non-bounds fractions. The wash and elution steps were performed as described previously for GFP co-immunoprecipitation.

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.