Nupage polyacrylamide gel

Protein lysates were harvested as described previously
[11 (link)]. 10 to 30 ug of lysate was separated using NuPage polyacrylamide gels (Life technologies, Mulgrave, VIC, Australia) prior to transfer to polyvinyl difluoride membranes. The membranes were incubated with the following primary antibodies: CDK1 (P34), CDK2 (M2), cyclin A (C-19), cyclin D1 (DCS-6), E2F1 (KH95) and MYC (9E10) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); BIM, cyclin E1, cyclin E2, phospho-CDK2 (Thr160), phospho-pRB (ser795) (Cell Signaling, Danvers, MA, USA); p21^Cip1/Waf1, p27^Kip1 and pRB from BD Pharmingen (San Diego, CA, USA), β-Actin (AC15) from Sigma (St Louis, MO, USA). The secondary antibodies were horseradish peroxidase-conjugated sheep anti-mouse or donkey anti-rabbit antibodies (Amersham, Rydelmere, NSW, Australia), and specific proteins were visualized by chemiluminescence (Perkin-Elmer, Rowville, VIC, Australia). Densitometry was performed using the software ImageJ.

Total protein from the livers were used for Western blot analysis as described previously [24 (link)]. Briefly, liver tissues were homogenized in RIPA buffer containing 5% mammalian proteinase inhibitor (Sigma-Aldrich, MO, USA). Protein concentrations were determined by BCA assay (Pierce, IL, USA). 50 μg protein samples were run on 4–12% NuPage polyacrylamide gels (Life Technologies NY, USA) and transferred to nitrocellulose membranes. After blocking with TBS containing 0.05% Tween-20, and 5% milk for 30 minutes, blots were incubated overnight at 4°C with rabbit anti-PARP antibody (Cell signaling Technology, MA, USA) [25 (link)] or mouse anti-GAPDH antibody (Fitzgerald, MA, USA) diluted in TBS-T containing 5% milk at 1:1000 dilution. The blots were washed with TBS, incubated with goat anti-rabbit IgG or Horse anti-mouse IgG antibody (Cell signaling Technology, MA, USA) tagged with peroxidase at 1∶5000 dilution in TBS containing 5% milk (Pierce, IL, USA) for 1 hr at room temperature. After 3×5 min washing with TBST, blots were incubated with freshly prepared SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, IL, USA) for 5 min and imaged using luminescent image analyzer (Image Quant LAS 4000, GE Healthcare Life Sciences, PA, USA). Densitometry analyses were performed using Image J software (National Institutes of Health, MD, USA).

The PBMC pellets were lysed on ice in lysis buffer. Following centrifugation to remove insoluble material, supernatants were collected and protein concentrations were determined using the Bradford method. Lysates were made up to equal concentrations with lithium dodecyl sulfate sample buffer added and subjected to SDS-polyacrylamide gel electrophoresis on 4–12% NuPAGE polyacrylamide gels (Novex, Life Technologies), then either stained using Coomassie Blue (SimplyBlue^TMSafe Stain, Invitrogen, CA, United States), or transferred to nitrocellulose membrane. Membranes were blocked in 5% (w/v) BSA, incubated with primary antibodies overnight at 4°C, and then Alexa-fluor secondary antibodies for 1 h at room temperature. Proteins were visualized using the Licor Odyssey. When probing for phospho Akt(Ser473), HRP secondary antibody was used, detection achieved with ECL and proteins visualized on film using different exposure times, typically between 10 s and 10 min.

The structural integrity of the Ad5 fiber proteins was assessed by Western blotting. An amount of 5×10⁹ vp/virus stock were run on ready-made 10% NuPAGE polyacrylamide gels (Invitrogen, Paisley, UK) by SDS-PAGE and transferred to Hybond-P nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK) by semidry blotting. Nitrocellulose membranes were treated with 5 ml of Pierce Miser antibody extender (Thermo Scientific) for 10 min and washing 7 times with distilled water. They were then blocked in 5% milk in Tris-buffered saline containing 0.05% TWEEN-20 and 0.05% Triton X-100 (TBS-T) overnight at 4°C. The membrane was incubated in primary anti-adenovirus fiber antibody 4D2 (1:2000) at 37°C for 1 hr, washed 5 times for 5 min in TBS-T, and incubated in antimouse IgG-HRP conjugate (1:2000; Insight Biotechnology Ltd., Wembley, UK) for 1 hr at room temperature. After washing a further 5 times for 5 min in TBS-T, the membrane was incubated for 5 min in SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific), and analyzed on GelDoc autoChemi camera (Ultra-Violet Products Ltd., Cambridge, UK).

Protein samples (20 µl) were separated on ready-made 10% Nu-PAGE polyacrylamide gels (Invitrogen) by SDS-PAGE and transferred to Hybond-P nitrocellulose (GE Life Science) by semi-dry blotting. Nitrocellulose membranes were prepared by treating with 5 ml Pierce MISER antibody extender (Fisher Scientific) for 10 min and washing 7 times with distilled water. They were then blocked in 5% milk in Tris-buffered saline containing 0.05% TWEEN-20 and 0.05% Triton X-100 (TBS-T-T) overnight at 4°C. The membranes were then incubated with primary antibodies diluted in 5% milk in TBS-T-T overnight at 4°C. They were then washed 5 times for 5 min in TBS-T-T, and incubated in anti-mouse IgG-HRP conjugate (Insight Biotechnology Ltd, Wembley, UK, 1∶5,000–1∶10,000) for 1 h at room temperature. After washing a further 5 times for 5 min in TBS-T-T, the membranes were incubated for 5 min in SuperSignal West Pico Chemiluminescent substrate (Fisher Scientific) before being exposed to Hyperfilm-MP film (GE Life Science, Little Chalfont, UK) for development. The blots were stripped in Pierce Stripper buffer (Fisher Scientific) for 10 min, washed 7 times in TBS-T-T, reblocked, and reprobed.