Las4000mini ccd camera apparatus

Cells were lysed on ice using Nonidet P-40 lysis buffer as fully detailed in [18 (link)]. Whole-cell extracts (WCE) were quantified using the Bradford protein assay (Bio-Rad, Hercules, CA, USA), resolved by SDS-PAGE and transferred to nitrocellulose membrane before analysis by immunoblot. Membranes were incubated with the following primary antibodies: anti-actin Cat #MAB1501 from Millipore (Burlington, MA, USA), anti-IRF9 Cat #610285 from BD Transduction Laboratories (San Jose, CA, USA), and anti-STAT1-P-Tyr701 Cat #9171, anti-STAT2-P-Tyr690 Cat #4441, anti-STAT1 Cat #9172, anti-STAT2 Cat #4594, all from Cell Signaling (Danvers, MA, USA), before further incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA or Jackson Immunoresearch Laboratories, West Grove, PA, USA). Antibodies were diluted in PBS containing 0.5% Tween and either 5% nonfat dry milk or BSA. Immunoreactive bands were visualized by enhanced chemiluminescence (Western Lightning Chemiluminescence Reagent Plus, Perkin-Elmer Life Sciences Waltham, MA, USA) using a LAS4000mini CCD camera apparatus (GE Healthcare, Mississauga, ON, Canada).

Nitrocellulose membranes were incubated with the following primary antibodies: anti-actin (Cat #A5441 from Sigma), anti-IFIT1 (Cat #14769S from Cell signaling, Danvers, MA, USA), anti-MAVS (Cat #A300-782AM from Bethyl, Montgomery, TX, USA), anti-Myc (Cat #M4439 from Sigma), and anti-NS3 (Cat #1847 from Virostat, Westbrook, ME, USA). After washes in PBS containing 0.5% Tween, membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (KPL, Gaithersburg, MD, USA or Jackson Immunoresearch Laboratories, West Grove, PA, USA). Antibodies were diluted in PBS containing 0.5% Tween and either 5% nonfat dry milk or BSA. Immunoreactive bands were visualized by enhanced chemiluminescence using Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences, Waltham, MA, USA). Chemiluminescence signal acquisition was performed using an LAS4000mini CCD camera apparatus (GE healthcare, Little Chalfant, UK).

Cell pellets were lysed on ice using Nonidet P-40 lysis buffer as previously described (14 (link)). Whole-cell extracts were quantified using the Bradford protein assay (Bio-Rad, Hercules, CA, USA), resolved by SDS-PAGE, and transferred to nitrocellulose membrane before analysis by immunoblot. Membranes were blocked in PBS containing 0.5% Tween (PBS-T) and 5% non-fat dry milk before incubation with the following primary antibodies: anti-actin (Cat #MAB1501, Millipore, Billerica, MA, USA), anti-tubulin (B-7, Cat #sc-5286, Santa-Cruz Biotechnology, Dallas, TX, USA) and anti-STAT1-phospho-Tyr701 (Cat #9171), anti-STAT3-phospho-Tyr705 (Cat #9145), anti-STAT1 (Cat #9172), and anti-STAT3 (Cat #4904) all from Cell Signaling Technology (Danvers, MA, USA). After washes in PBS-T, membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (KPL, Milford, MA, USA, or Jackson Immunoresearch Laboratories, West Grove, PA, USA). Antibodies were diluted in PBS containing 0.5% Tween and either 5% non-fat dry milk or BSA. After washes in PBS-T, immunoreactive bands were visualized by enhanced chemiluminescence (Western Lightning Chemiluminescence Reagent Plus, Perkin-Elmer Life Sciences, Waltham, MA, USA) using a LAS4000mini CCD camera apparatus (GE Healthcare, Little Chalfont, UK).