Hrp labelled secondary antibody

Protein lysates were obtained by lysis of cultured cells in RIPA buffer (Sigma-Aldrich) including protease inhibitor cocktail (Complete EDTA-free, Roche). Protein samples were denaturated in Laemmli buffer (60 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulphate, 10% glycerol, 5% β-mercaptoethanol and 0.01% bromphenol blue) by heating at 95°C for 10 min and were subsequently loaded on 4%-15% Mini-PROTEAN TGX Precast Gels (BioRAD). After separation, proteins were transferred to a nitrocellulose membrane (Amersham, Freiburg, Germany) and immunoblotted with the anti-TROY-antibody (dilution 1:500; ab137080; Abcam) and an anti-β-actin-antibody (1∶30,000; clone AC-15; Sigma-Aldrich) to ensure equal loading amounts. Membrane bound HRP labelled secondary antibodies (DakoCytomation, Glostrup, Denmark) were detected by enhanced chemiluminescence using the ECL system (Amersham).

Vero cells seeded in 6-well plates were mock infected or infected with undiluted IBV (MOI 0.005). At the indicated time points, cells were washed once with cold PBS and lysed in 1× sample buffer (Biorad) containing DTT. Cell lysates were heated to 95 °C for 3 min and briefly sonicated. Proteins were separated on a 4%–20% Bis-Tris gel (Biorad) and transferred onto nitrocellulose membranes. These were blocked in 0.5% BSA or 5% milk in TBS-Tween (TBS-T), then incubated with primary antibody diluted in blocking buffer. Following three washes in TBS-T, membranes were incubated with HRP labelled secondary antibodies (Dako), diluted in blocking buffer. After three further washes in TBS-T, blots are incubated chemiluminescence substrate using the Clarity Western ECL Substrate (Bio-Rad). Labelled protein bands were visualized, using a Vilber imaging system. Anti-IBV was diluted 1:1000, anti-eIF2α (Cell Signaling Technologies) was diluted 1:1000, anti-eIF2α-p (Cell Signaling Technologies) was diluted 1:2000 and anti-GAPDH (Invitrogen) was diluted 1:10000.

Skeletal muscle and liver samples were powdered with ultrasonic cell crush. Each homogenate (10 µL) was mixed with an equal amount of standard SDS sample loading buffer containing 0.25% bromophenol blue, and subjected to SDS-PAGE electrophoresis. After that, protein was transferred onto PVDF membrane by electroblotting. The membranes were then treated sequentially with blocking solution, and then with anti-GLUT-4, anti-Akt-2, anti-PTP-1B, anti-SOCS-3, or anti-JNK antibody (RD Inc, Minneapolis, MN). After washing, membranes were incubated with HRP-labelled secondary antibody (DAKO, Carpinteria, CA). Finally, electrochemiluminescence (ECL) was used to visualize the bands.