Mouse β actin antibody

Islets were lysed in buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 4 mM EDTA, 1% Triton X-100, 10% glycerol, 50 mM NaF and 10 mM sodium pyrophosphate, supplemented with 0.1 mM phenylmethylsul- fonyl fluoride, 5 ng/ml leupeptin, 1 μg/ml aprotinin and 2 mM Na₃VO₄ for 1 h at 4 °C. After centrifugation (14 000 g for 30 min) supernatants were collected and protein concentration measured by Bradford assay. Equal protein amount (20 μg) from the islets lysates were separated by SDS-PAGE. Membranes were probed with primary antibody against IkB-α (sc-371, Santa Cruz Biotechnology Inc., Dallas, TX, USA). Mouse β-actin antibody (Sigma-Aldrich) was used to normalize protein levels. Band intensities were quantified by ImageJ software (1.48v, National Health Institute, NIH, Bethesda, MD, USA).

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.