Ac 15

Samples were made up in dissociation buffer (1× dissociation buffer (100 mm Tris-HCl, 2% (w/v) SDS, 10% (v/v) glycerol, 100 mm DTT, 0.02% (w/v) bromphenol blue, pH 6.8) and heated at 95 °C for 5 min. Proteins were resolved by SDS-PAGE on 7–17% acrylamide Tris-glycine gels and then transferred to Hybond polyvinylidene difluoride membranes (GE Healthcare). Following electrotransfer, the membranes were blocked for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% (w/v) nonfat milk and then incubated with primary antibody overnight at 4 °C. Antigens were probed using the following primary antibodies: anti-APP (22C11, Millipore), SAF32 (anti-PrP N terminus, Cayman Chemical), 8H4 (anti-PrP residues 175–185), anti-ADAM10 antibody (Abcam), 6E10 (anti-Aβ(1–17), Merck Biosciences), AC15 (anti-β-actin) and synapsin 1 (Sigma), 2B3 (anti-human sAPPα, Immuno-Biological Laboratories), and anti-phospho-Src family kinase (Tyr-416; Cell Signaling Technology). Primary antibodies were detected by incubation with horseradish peroxidase–conjugated secondary antibody, both in PBST containing 2% BSA. Bound horseradish peroxidase conjugates were visualized using the ECL® detection system with a Syngene Gbox XT4 (Syngene). Densitometric analysis was performed using Genetools analysis software (Syngene).

Primary antibodies specific for HBV proteins included anti-HBV core protein mouse monoclonal antibody (MAb) 8C9-11 (72 (link)), anti-HBV core protein mouse MAb sc-23945 (Santa Cruz), both recognizing linear epitopes within the HBV core protein, and HBV capsid/capsid-intermediate-specific rabbit polyclonal antibody (PAb) B0586 (Dako). Primary antibodies used for epitope tags and cellular targets included anti-HA rat MAb 3F10 (Core Facility for Monoclonal Antibodies, Helmholtz Zentrum München), mouse MAb against the His₆ tag (Clontech), anti-PML protein mouse MAb sc-966 (Santa Cruz), polyclonal rabbit anti-PML protein PAb NB100-59787 (Novus Biologicals), polyclonal rabbit anti-PML protein ab72137 (Abcam), monoclonal mouse anti-SUMO2/3 ab81371 (Abcam), and anti-beta-actin mouse MAb AC-15 (Sigma-Aldrich). Secondary antibodies conjugated to Alexa Fluor 488 were purchased from Thermo Scientific, and secondary antibodies coupled to horseradish peroxidase or Alexa Fluor 647 were anti-rabbit IgG, anti-mouse IgG, anti-mouse light chain IgG, and anti-rat IgG (all from Dianova).

The following antibodies were used:
Rabbit monoclonal anti-EGFR (ab52894, Abcam, Cambridge, UK); mouse monoclonal anti-EGFR (sc-120, Santa Cruz Biotechnology, USA); mouse monoclonal anti-EGFRvIII (L8A4, Absolute Antibodies, UK); rabbit polyclonal anti-EGFR (ab5652, Abcam, UK); mouse monoclonal anti-HO-1 (ab13248, Abcam, UK); mouse monoclonal anti-β-actin (AC-15, Sigma-Aldrich, USA); IRDye^® 800CW goat anti-mouse IgG (827-08364, LI-COR Biotechnology, Germany); IRDye^® 680RD goat anti-rabbit IgG (926-68071, LI-COR Biotechnology, Germany).

Protein samples (20μg) were electrophoresed on 4-12% Novex Tris-Glycine pre-cast gels (Life Technologies, California, USA) using non-reducing conditions in 1x MOPS running buffer (Astral Scientific, NSW, AUS) and transferred to Hybond™-C Extra Nitrocellulose membrane (GE Healthcare). Membranes were incubated overnight in TTBS (Tris-buffered saline with 0.1%Tween-20) at 4°C, blocked for 30min in 5% skim milk powder in TTBS. Anti-CD9 primary antibody (clone1AA2 4μg/mL; made in-house, Ashman Laboratory, AUS) or anti-β-actin (1:20000; AC-15; Sigma-Aldrich) were detected with HRPD-conjugated goat anti-mouse secondary antibody (1:5000, Bio-Rad (72-1011), California, USA) and enhanced chemiluminescence (25mL TBS, 2.5mM Luminal, 0.4mM PCA and 2.6mM hydrogen peroxide) with the Fujifilm LAS4000 (Fujifilm, Tokyo, Japan).