Polyclonal rabbit anti rat caspase 3

Twenty µg of protein was electrophoresed as follows: The total proteins were homogenized with a prechilled mortar and pestle in extraction buffer consisting of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 µg/mL pepstatin A, and 10 µg/mL aprotinin. The homogenate was centrifuged at 12,000 rpm for 12 min at 4 °C, the supernatant was collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Antibodies raised against the monoclonal mouse anti-human PARP (Promega, Madison, WI, USA), polyclonal rabbit anti-rat caspase 3 (Chemicon, CA, USA), primary antibody Bcl-2 (Santa Cruz, Dallas, TX, USA), Bax (Abcam, Cambridge, UK), rabbit polyclonal EPO (Abcam, Cambridge, UK), and monoclonal mouse anti-mouse β-actin (Sigma, Saint Louis, MI, USA) were used at 1:400. The membranes were incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (Biolegend, San Diego, CA, USA) for 1 h, washed in TBST, and imaged with Image Quant^TM LAS 4000 (GE, Boston, MA, USA).

Twenty µg of protein was electrophoresed as follows: The total proteins were homogenized with a prechilled mortar and pestle in extraction buffer consisting of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethyl sulfonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 2% β-mercaptoethanol, 10 µg/mL pepstatin A, and 10 µg/mL aprotinin. The homogenate was centrifuged at 12,000 rpm for 12 min at 4 °C, the supernatant was collected, and the protein concentrations were determined by BioRad Protein Assay (BioRad Laboratories). Antibodies raised against the monoclonal mouse anti-human PARP (Promega, Madison, WI, USA), polyclonal rabbit anti-rat caspase 3 (Chemicon, CA, USA), primary antibody Bcl-2 (Santa Cruz, Dallas, TX, USA), Bax (Abcam, Cambridge, UK), rabbit polyclonal EPO (Abcam, Cambridge, UK), and monoclonal mouse anti-mouse β-actin (Sigma, Saint Louis, MI, USA) were used at 1:400. The membranes were incubated with goat anti-rabbit or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (Biolegend, San Diego, CA, USA) for 1 h, washed in TBST, and imaged with Image Quant^TM LAS 4000 (GE, Boston, MA, USA).