Murine antibody

Whole-cell lysates were made from monolayers of cultured cells in the exponential phase of growth. Cells were subjected to various treatment protocols (as indicated in the figure legends) after seeding into tissue culture flasks at 30–40% confluence. Cells were harvested at appropriate time points using a non-enzymatic cell dissociation fluid, washed twice in PBS and resuspended in RIPA buffer. Whole-cell lysate protein was loaded onto SDS-PAGE gels, electrophoresed, and Western transferred to a PVDF membrane. Probing of the membranes for GAPDH levels to act as loading controls was carried out by membrane stripping with 1 mM sodium azide in PBS for 1 h, followed by incubation using a murine antibody (Sigma Aldrich, St. Louis, MI, USA). Visualisation was carried out using alkaline phosphatase-conjugated secondary antibody (WesternBreeze^®, Life technologies, Paisley, UK) with chemiluminescent substrate (CDP Star^®, Invitrogen, Carlsbad, CA, USA). Protein expression levels were quantified by use of densitometry using chemiluminescent film, and processing of the scanned images as JPEG files was performed using Quantiscan 3.0 software (Biosoft^®). Band intensity was normalised to GAPDH and was expressed relative to MCF-7/T47D controls (values shown are the means ± SD of three independent experiments, with p-values shown in the legends).

For the binding assay, HaCaT cells were preincubated for 1 h with the original or tannin-free (Section 2.6) extracts or DMSO and were then infected for 15 min with 500 HPV pseudovirions per cell. Cells were washed five times with PBS and collected in SDS sample buffer for Western blotting. Cell-bound HPV16 particles were stained with anti-L1 antibody 312F [62] (link). β-Actin (loading control) was stained using a murine antibody (Sigma-Aldrich) and relative band intensities were quantified densitometrically.
For the HPV capsid disassembly assay, HaCaT cells were grown on coverslips and treated with 20 µg/ml of the extracts for 1 h before HPV16 pseudovirion infection for 7 h at 37°C. Cells were fixed with methanol and stained with mouse anti-L1 antibody (33L1-7) as described previously [63] (link), [64] (link). L1-7 recognizes an epitope located inside of the pseudovirion capsid and is only accessible after uncoating [65] . Fluorescence was recorded using a Zeiss Axiovert 200 M microscope. For quantification, the relative amount of internalized particles was determined based on the L1-7-positive pixels relative to the cell nucleus signal (DNA/Hoechst 33342-positive pixels) out of 100 randomly selected cells from two independent experiments. A threshold value was set to exclude background.