Sepharose 4B gel chromatography of media constituents from HPV-exposed HaCaT cells.

<p>Sepharose 4B chromatography was performed on released components in the CM of HaCaT cells exposed to HPV16 PsV for 4 h; see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002519#ppat.1002519.s002" target="_blank">Figure S2</a>. (A) The void volume (HMW) fraction was divided into four parts that were untreated or incubated with 1 U heparinase III for 2 h at 37°C then solubilized in 6× sample buffer and incubated at 25°C or boiled for 7 min before SDS-PAGE and L1-immunoblot analysis. Separate lanes shown are from the same exposure of the same film. (B) Non-reducing SDS-PAGE of void volume Sepharose 4B fractions from released components in the CM of HaCaT cells mock-exposed or HPV16-exposed for 24 h. Immunoblot analysis was done to detect L1, amphiregulin (AREG), HB-EGF, EGF, HS and syndecan-1 (snd-1). (C) Non-reducing SDS-PAGE of released components following IP of HPV16 from CM of HaCaT cells mock- or HPV16-exposed. HPV16 exposed cells were allowed to bind virus at 4°C for 1 h, washed to remove unbound particles, then shifted to 37°C for 6 h or 24 h. CM were subjected to IP for HPV16 using affinity purified polyclonal anti-HPV16 VLP antibody covalently attached to magnetic beads. Immunoblot analysis was performed to detect HB-EGF, EGF, and syndecan-1.