Xpert protease inhibitor cocktail solution 100

Cells were lysed in lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 5 mM EDTA, 0.1% NP40, and Xpert protease inhibitor cocktail Solution 100× (catalog no. p3100-010; GenDEPOT, Barker, TX, USA). Protein concentration was determined by Bradford assay. A 10–12% SDS-polyacrylamide gel was run under standard electrophoresis conditions, with 30 µg of total protein loaded in each lane. Separated proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA) at 100 V for 100 min, and incubated with primary antibodies in blocking solution (3% BSA). Proteins were visualized using the Western blotting luminol reagent (catalog no. sc-2048; Santa Cruz, Dallas, TX, USA). The following antibodies were used: mouse anti-NS5B; 5B-3B1 (Enzo Life Science; Switzerland), mouse anti-NS3; sc-69938, mouse anti-NS4B; sc-52416, mouse anti-NS5A; sc-65458, goat anti-Lamin B; sc-6216, mouse anti-α-Actinin; sc-17829, mouse anti-β-actin; sc-47778, mouse anti-TRAF6; sc-8409 (Santa-Cruz, Dallas, TX, USA), rabbit anti-NF-kB(P65); 8242s, rabbit anti-p62; 8025s, and rabbit anti-LC3B; 2775s (Cell signalling, Danvers, MA, USA). All first antibody dilution was 1:1,000. Secondary antibodies used goat anti-mouse IgG; M32607, goat anti-rabbit IgG; 31460 (Invitrogen, Waltham, MA, USA), rabbit anti-goat IgG; SA007-500 (GenDEPOT, Barker, TX, USA). All secondary antibody dilution was 1:10,000.

Treated or non-treated cells were lysed in lysis buffer containing 50 mM Tris-Cl, 150 mM NaCl, 12 mM sodium deoxycholate, 1% Triton x-100, 0.1% SDS and Xpert protease inhibitor cocktail Solution 100× (catalog no. p3100-010; GenDEPOT, Barker, TX, USA), and cell lysates were cleared by centrifugation at 14,000 rpm for 15 min at 4 °C. The lysates (1 mg) were incubated with anti-TRAF6 (4 ug) for 24 h at 4 °C. Protein-A/G-conjugated agarose beads were added, followed by incubation for 4 h at 4 °C. Beads were washed three times with 1X TBST. Immunopellets were boiled with SDS-PAGE sample buffer and resolved by electrophoresis. The following antibodies were used: mouse anti-TRAF6; sc-8409, mouse anti-β-actin; sc-47778 (Santa-Cruz, Dallas, TX, USA), and rabbit anti-p62; 8025s (Cell signalling, Danvers, MA, USA).