Rabbit monoclonal anti mcl 1 antibody

The cytosolic extracts were obtained using a commercial kit (Active Motif, Carlsbad, CA, USA) following the manufacturer’s protocol. For immunoblotting, the following antibodies were used: mouse monoclonal anti-caspase 3 antibody (Active Motif, Carlsbad, CA, USA), rabbit monoclonal anti-Mcl-1 antibody (Cell Signaling, Leiden, Netherlands), rabbit monoclonal anti-Bcl-2 antibody (Cell Signaling, Leiden, Netherlands), and mouse monoclonal anti-β-actin (Sigma-Aldrich, Milan, Italy) as internal control. The secondary antibodies peroxidase conjugated were anti-mouse (BD Pharmingen, Franklin Lakes, NJ, USA) and anti-rabbit (Cell Signaling). Protein bands were quantified using ImageJ software (U.S. National Institutes of Health, Bethesda, MA, USA).

After appropriate treatments cells were collected, counted and 1 million of cells were re-suspended in 2X Laemmli Buffer (4% SDS, 20% Glycerol, 10% 2-Mercaptoethanol, 0.004% Bromophenol Blue and 0.125M Tris HCL). Twenty microliter of protein were then subjected to SDS-polyacrylamide gel electrophoresis in 12% polyacrylamide gels followed by protein transfer to polyvinyl difluoride (PVDF) membranes (Thermo Scientific) and immunoblotted with mouse monoclonal anti Bcl2 antibody (Dako, Carpentaria, CA), rabbit monoclonal anti Mcl-1 antibody, rabbit monoclonal anti-AMPKα, rabbit monoclonal anti-phospho(Thr172)AMPKα (All from Cell Signaling Technology), and mouse monoclonal anti-β-actin (Sigma-Aldrich). Signals were detected by immunofluorescence, using fluorochromes conjugated anti-rabbit and anti-mouse secondary antibodies and the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA).