Pierce enhanced chemilumescent ecl western blotting substrate

293 and A549 cells were seeded at 1x10⁶ and 0.8x10⁶ cells per 35mm dish, respectively. Next day, the cells were infected for 1 h at a multiplicity of infection (MOI) of 10 with AdEmpty or AdFAST-HA. Following a 48 h incubation, whole cell lysates were collected using the cell lysis buffer as described by Lieber et al. [27 (link)]. Samples were separated by 15% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The resulting membrane was probed with a mouse anti-HA tag monoclonal antibody (1:1000, Cell Signaling #2367) and goat anti-mouse antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170–6516). To confirm Ad replication, the membrane was reprobed for Ad5 fibre (1:20 000 mouse anti-fibre monoclonal antibody, clone 4D2, Neomarkers). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit anti-α-tubulin antibody, AbCam #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170–6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific). Densitometry was performed using AlphaEaseFC (Alpha Innotech).

4T1 cells were seeded at 1x10⁶ cells per 35 mm dish. Next day, the cells were infected for 1 h at the indicated MOI of Ad virus. Following a 48 or 72 h incubation, whole-cell lysates were collected using 2 × SDS/PAGE protein loading buffer (62.5 mm Tris HCl pH 6.8, 25% glycerol, 2% SDS, 0.01% bromophenol blue, 5% β-mercaptoethanol). Samples were boiled for 5 min, separated by electrophoresis on a 15% SDS-polyacrylamide gel, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Etobicoke, ON, Canada). The resulting membrane was probed with a mouse HA tag monoclonal antibody (1:10 000, Cell Signaling (Beverly, MA, USA) #2367), rabbit cleaved caspase-3 monoclonal antibody (1:1000, Cell Signaling #9664) or full-length caspase-3 antibody (1:1000, Cell Signaling #9662) and 1:5000 goat anti-rabbit IgG (Bio-Rad, Mississauga, ON, Canada, #170-6515) or goat anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (1:10 000, Bio-Rad #170-6516). The membrane was also probed with antibody to α-tubulin to confirm equal loading (1:5000 rabbit α-tubulin antibody, AbCam (Toronto, ON, Canada) #ab15246, 1:5000 goat anti-rabbit IgG conjugated to HRP, Bio-Rad #170-6515). Blots were developed using the Pierce Enhanced Chemilumescent (ECL) Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA).