Nupage novex pre cast 4 12 bis tris gradient gels

Cells were lysed using radioimmune precipitation assay (RIPA) buffer containing 1% NP-40, 0.1% SDS, 10 mM Tris–HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl and protease inhibitor cocktail (Roche), and sonification was performed subsequently. Proteins were separated by SDS–PAGE or NuPAGE Novex pre-cast 4–12% Bis–Tris gradient gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. The primary antibodies used were: anti-STAT1, anti-phospho-STAT1 (Tyr701) anti-STAT2 and anti-phospho-STAT2 (Cell Signaling Technology, Inc., Beverly, MA), mouse anti-HCV core, (Thermoscientific), and mouse anti-actin (Sigma Life Science and Biochemicals, St. Louis, MO). Secondary antibodies were: HRP-conjugated ECL donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ). The ECL Western Blotting Detection Kit (Amersham Biosciences, Piscataway, NJ) was used to detect the chemiluminescent signals.

Cells were lysed using radioimmune precipitation assay (RIPA) buffer containing 1% NP-40, 0.1% SDS, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150 mM NaCl and protease inhibitor cocktail (Roche), followed by sonification. Proteins were separated by SDS-PAGE with NuPAGE Novex pre-cast 4–12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. The primary antibodies used were anti-STAT1, anti-phospho-STAT1, anti-STAT2, anti-phospho-STAT2, anti-MAVS, anti-TRIF, anti-PKR, anti-IRF3 (Cell Signaling Technology, Inc., Beverly, MA), anti-HCV core, (Thermoscientific), anti-HCV NS3, anti-HCV NS5A and anti-NS5B (Virogen, Watertown, MA), anti-ISG15, anti-MxA and anti-TRIF (Abcam), anti-TRF3 (Santa Cruz), anti-phospho-IRF3 (Origene) and anti-β-actin (Sigma Life Science and Biochemicals, St. Louis, MO). DENV-2 NS3 was detected by a specific monoclonal antibody against NS3 (Yao-Hong Biotechnology). Secondary antibodies were HRP-conjugated ECL donkey anti-rabbit IgG and HRP-conjugated ECL sheep anti-mouse IgG (Amersham Biosciences, Piscataway, NJ). The ECL Western Blotting Detection Kit (Amersham Biosciences, Piscataway, NJ) was employed to detect chemiluminescent signals. Immunoblots shown in each figure are representative of three independent experiments. Densitometry was performed with ImageJ software. Student's t-test was used as statistical test.

For total parasite protein extraction, 7-week male worms were homogenised in lysis buffer (20 mM KH₂PO₄, pH 7.4 and 0.1% v/v Triton X-100) using a Tissue Lyser (Qiagen). Soluble yeast proteins (described in section 2.9) and parasite extracts were electrophoresed on NuPAGE Novex pre-cast 4–12% Bis-Tris gradient gels (Invitrogen) and transferred onto polyvinylidene fluoride (PVDF) membranes in NuPAGE transfer buffer (Invitrogen). Membranes were blocked in PBS/Tween-20 (0.3% v/v) containing 5% skimmed milk powder (blocking buffer) overnight at 4 °C before incubation with antibodies. In the case of yeast blots, a GAL4-BD-horseradish peroxidase (HRP)-linked antibody (Santa Cruz Biotechnology) was used (1:1000 dilution for 1 h) in PBS/Tween-20 (0.3% v/v) containing 1% skimmed milk powder. In the case of parasite blots, the membrane was incubated with anti-rSmVAL6 (1:600 dilution in blocking buffer) for 3 h (Rofatto et al., 2012 (link)). Following 3 × 10 min washes in PBS/Tween-20 (0.3% v/v), the membrane was incubated with goat anti-mouse IgG conjugated HRP (1:2000 in blocking buffer) for 1 h. All blots received a final 3× wash in PBS/Tween-20 (0.3% v/v) and were subsequently developed using ECL-Plus reagent (GE Healthcare).