Monoclonal antibodies to the bkv major capsid protein vp1

Cell extracts were prepared using RIPA lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA) pH 8.0, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and proteinase inhibitor (cOmplete ULTRA, Roche, Basel, Switzerland). Lysates were incubated on ice for 30 minutes and then clarified by centrifugation. Total protein was measured using a micro BCA protein assay kit (ThermoFisher Scientific). Protein lysates (30 μg) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), blocked with 5% milk in 0.1% TBST (0.1% Tween 20, 20 mM Tris, 150 mM NaCl), and incubated at 4 °C overnight with monoclonal antibodies to the BKV major capsid protein VP1 (Santa Cruz Biotech) at 1:250 dilution. A synaptopodin antibody (Santa Cruz Biotech) was used at 1:250 dilution and the GAPDH antibody (Santa Cruz Biotech) was used at 1:3000 dilution. Membranes were washed five times in 0.1% TBST and incubated for one hour with a corresponding secondary antibody conjugated with HRP (ThermoFisher Scientific) at a dilution of 1:50,000. Immunoreactive bands were detected with WesternBright ECL (Advansta, San Jose, CA) following exposure to X-ray film.

Immunofluorescent staining was performed as previously described [Alcendor 2017] in chamber slide cultures containing mock and BKV-infected GVU cells (podocytes, mesangial cells, and glomerular endothelial cells). Briefly, cells were washed twice with PBS, pH 7.4, air- dried, and fixed in absolute methanol for 20 min at −20 °C. Next, cells were air-dried for 10 min, hydrated in Tris-buffered saline (TBS) (pH 7.6) for 10 minutes, and incubated separately for 1 h with monoclonal antibodies to the BKV major capsid protein VP1 (Santa Cruz Biotech, Temecula, CA, USA), von Willebrand factor (Santa Cruz Biotech), nephrin (Santa Cruz Biotech), and SV40 Large T-Antigen (Abcam), all at a dilution 1:50 in PBS pH 7.4 [30 (link)].