Hrp labeled anti mouse antibody

One million BON and NCI-295R cells per well were seeded in 6-well plates and treated for 2 and 6 h, respectively, with TNFα on the following day. Cells were lysed in the RIPA buffer containing Complete Mini Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The protein concentration was measured using the BCA kit (Thermo Fisher) with the PowerWave340 plate reader (Biotek, Winooski, VT, USA). With 10 µg of proteins loaded per well on the 3.9–20% gel, PAGE was performed for 2 h 30 min at constant voltage of 30 mA per gel in Mini-PROTEAN Tetra Vertical Electrophoresis Cell (Bio-Rad, Hercules, CA, USA).
The eBlot transfer system (Genscript, Piscataway Township, NJ, USA) was used for protein transfer on NC membranes. After blocking with NET-G buffer for 1 h, the membranes were incubated with LC3B antibody (Abcam) at +4 °C overnight, washed, and incubated with the HRP-labeled anti-rabbit antibody (GE Healthcare, Chicago, IL, USA) for 1 h at RT. For visualization, Lumi-Light ECL (Roche) and the LAS3000 imager (Fujifilm, Minato, Tokyo, Japan) were used. Subsequently, membranes were washed, blocked with NET-G, and incubated with mouse β-actin antibody (Sigma-Aldrich) at +4 °C overnight. As the secondary antibody, HRP-labeled anti-mouse antibody (GE Healthcare) was used. β-actin was visualized with Lumi-Light ECL in LAS3000.

A total of 0.7 m BON and 1 m NCI-295R cells per well were seeded in 6-well plates and treated for 24 h with 0.1 µg/mL TNFα starting the following day. Cells were lysed in RIPA buffer containing Complete Mini Protease Inhibitor Cocktail (Roche). The protein concentration was measured using the BCA kit (Thermo Fisher) with the Tecan Sunrise plate reader (Tecan, Männedorf, Switzerland). A sample with 15 µg of proteins was loaded on Any kD™ Mini-PROTEAN^® TGX Stain-Free™ Protein Gels (Bio-Rad, Hercules, CA, USA) and run at constant 100 V until the dye front reached the reference line of the gel. For protein transfer, PVDF membranes and Trans-Blot Semi-Dry Transfer Cell (Bio-Rad) were used. Membranes were cut and after blocking with Blotting-Grade Blocker (Bio-Rad), the upper halves were incubated with mouse anti-MVP antibody (Abcam, Cambridge, UK) and the lower halves were incubated with mouse β-actin antibody (Sigma-Aldrich) at +4 °C overnight, washed, and incubated with HRP-labeled anti-mouse antibody (GE Healthcare). As the ECL substrate, Western Lightning Plus (PerkinElmer, Waltham, MA, USA) was used. The luminescence signal was captured with the Hyperfilm ECL (GE Healthcare).