About 6 × 105 HaCaT-GFP-BAP cells were seeded in 24-well plates. The following day, cells were infected with 600, 200, or 67 ng L1/well of HPV16 WT or SSTP212AAAA L2-BirA PsVs. 20 h p.i., samples were rinsed with PBS, surface virus was removed by an alkaline PBS (pH10.6) wash followed by another PBS rinse, and samples were processed for reducing SDS-PAGE followed by Western blot analysis40 (link). L2 was detected by mouse monoclonal anti-L2 (K4) (a kind gift of Martin Müller) and GFP by rabbit anti-GFP (Takara Bio Clonetech). IR-Dye conjugated secondary antibodies anti-mouse and anti-rabbit (LI-COR Biosciences GmbH) were used and blots were imaged on the LI-COR Odyssey Infrared Imaging System.
For inhibition studies, the viral inoculum (2 × 108 viral genome equivalents/well) was not removed from cells, and luciferase assays were performed at 24 h p.i. DMSO carrier or PLK1 inhibitors BI2536 and BI6727 (each at 100 nM final concentration) were added at the time of infection and left for the duration.
For inhibition studies, the viral inoculum (2 × 108 viral genome equivalents/well) was not removed from cells, and luciferase assays were performed at 24 h p.i. DMSO carrier or PLK1 inhibitors BI2536 and BI6727 (each at 100 nM final concentration) were added at the time of infection and left for the duration.
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