Immunoblotting Characterization of Viral Proteins

Proteins were extracted from tissue sections as previously described [36 (link)] and quantified using the BCA protein assay kit (Pierce, UK), before being separated on 4–12% gradient polyacrylamide-SDS-Tris-Tricine denaturing gel (Invitrogen, UK) and transferred onto PVDF membranes (Bio-Rad). After transfer, membranes were blocked for 1 hour at room temperature in 1% milk in PBS-T (PBS, 0.1% tween20). Blots were then incubated overnight at 4°C with the appropriate primary antibody diluted in 1% milk PBS-T. Primary antibodies used were anti-E2 rabbit polyclonal antisera [35 (link)], anti-tubulin clone B512 (Sigma, UK), anti-HPV16L1 (CAMVIR-1, Santa Cruz, USA), anti-GFP clone B-2 (Santa, Cruz), anti-16E1^E4 antibody TVG 405 or anti-GAPDH clone 374 (Chemicon, UK), followed by the appropriate HRP-conjugated secondary antibody (GE Healthcare, UK), and detection using ECL, or ECL plus kits (GE Healthcare, UK) or by the appropriate IRDye 800CW fluorescent secondary antibody (Licor, UK) followed by detection using an Odissey imaging system (Licor, UK).