Horseradish peroxidase conjugated anti mouse secondary antibody

To analyse the protein levels of p53, caspase-3, GRP78, LC3 and p62, HepG2 cells were treated with P. tomentosa compounds (1 or 2 μM) or with DMSO for the indicated times; then, Western blot analyses were performed. Briefly, cells were lysed in RIPA buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1% sodium dodecyl sulphate, 1% triton X-100, 1 mM orthovanadate, and a cocktail of inhibitors (Sigma-Aldrich, Milan, Italy)). After separation on a sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transfer to a poly-vinylidene fluoride membrane (Euroclone, Milan, Italy), the following primary antibodies were used, at a dilution of 1:1000 in tris-buffered saline containing 1% non-fat dry milk, overnight at 4 °C: mouse anti-p53 antibody, mouse anti-MAP LC3 β antibody, mouse anti-p62 antibody (DBA, Milan, Italy), mouse anti-GRP78 antibody, and mouse anti-caspase 3 antibody (Thermo Fisher Scientific, Milan, Italy). For normalisation, a mouse anti-GAPDH antibody (Microtech, Naples, Italy) was used. A horseradish-peroxidase-conjugated anti-mouse secondary antibody (Bio-Rad laboratories S.r.l, Milan, Italy) was used for 1 h; finally, immunocomplexes were revealed using a chemiluminescence detection kit (Microtech, Naples, Italy) according to the manufacturer’s instructions.

The cells were lysed in Cell Lysis Buffer (Cell Signaling Technology, Delaware, CA, USA) containing a 1x Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The protein content was measured with a protein assay kit (Pierce, Hercules, CA, USA). Protein samples (15 μg), together with a protein marker (Precision Plus Protein Western C Standards; Bio-Rad), were separated on 12% Mini-Protean TGX gels (Bio-Rad, Richmond, CA, USA) for 30 min at 200 V. The separated gels were transferred to a polyvinylidene fluoride (PVDF) membrane for 3 min using the Trans-Blot Turbo Transfer system (Bio-Rad) with Trans-Blot Transfer Packs. Western blots with Runx2), ALP, OSX, and β-actin (ACTB) were processed on the iBind Western System (Life Technologies, Carlsbad, CA, USA) as specified by the antibody manufacturer (anti- Runx2) [Abcam, Tokyo, Japan], anti-ALP [Abcam], anti-Sp7/osterix [OSX; Abcam], and anti- ACTB [Bio-Rad] primary antibodies; and horseradish peroxidase-conjugated anti-mouse secondary antibody [Bio-Rad]). The incubated membranes were developed using an enhanced chemiluminescence system (SignalFire Plus ECL Reagent; Cell Signaling Technology). The band density was quantified using the NIH-Image J software and normalized to that of ACTB on day 0.