Irdye 800cw fluorescent secondary antibody

Proteins were extracted from cells using RIPA buffer and quantified using the BCA protein assay kit (Pierce), before being separated on 4–12% gradient polyacrylamide-SDS-Tris-Tricine denaturing gel (Invitrogen) and transferred onto PVDF membranes (IPFL00010, Merck). After transfer, membranes were blocked for 1 hour at room temperature in 5% milk in TBS. Blots were then incubated overnight at 4°C with appropriate primary antibody diluted in 5% milk in TBS. This is followed by incubating with appropriate IRDye 800cW fluorescent secondary antibody (Licor) for an hour at room temperature. Protein bands were detected with Odissey imaging system (Licor). Primary antibodies used in this study are listed in S3 Table.

Proteins were extracted from cells using radioimmunoprecipitation assay (RIPA) buffer and quantified using the bicinchoninic acid (BCA) protein assay kit (Pierce, MA, USA), before being separated on 4 to 12% gradient polyacrylamide SDS-Tris-Tricine denaturing gel (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). After transfer, membranes were blocked for 1 h at room temperature in 1% milk in PBS-T (phosphate-buffered saline plus 0.1% Tween 20). Blots were then incubated overnight at 4°C with the appropriate primary antibody diluted in 1% milk in PBS-T. The primary antibodies used were anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (clone MAB374; Millipore, MA, USA), anti-MAML1 (clone D3E9; Cell Signaling Technology, MA, USA), and anti-hemagglutinin (anti-HA) (clone ab130275; Abcam, Cambridge, United Kingdom). Membranes were then incubated with IR dye (800CW) fluorescent secondary antibody (LI-COR, NE, USA) for 1 h at room temperature and then washed. Finally, proteins were detected using the Odyssey imaging system (LI-COR, NE, USA).

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).