Rabbit monoclonal antibody to gapdh

Tissues were homogenized in PBS and then lysed with 5× SDS-PAGE sample buffer by sonication for 5 minutes and boiling for 10 minutes. Samples were separated by SDS-PAGE and transferred to PVDF membranes (GE Healthcare). Membranes were blocked with 5% skim milk for 1 hour, followed by incubation with the primary antibodies at 4°C overnight. Primary antibodies used were as follows: rabbit polyclonal antibody against Iqcg (1∶500 dilution, generated by this study), rabbit monoclonal antibody to GAPDH (1∶1000 dilution, code #2118, Cell Signaling Technology), mouse monoclonal antibody to Calmodulin (1∶500 dilution, code 05-173, Millipore). After washed with TBST buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween-20) for three times, membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology) for 1 hour. After washed with TBST for three times, specific proteins bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore) with a Fujifilm LAS-4000 system.

Whole-cell lysates were obtained by lysing equal numbers of cells with 3× laemmli buffer (30% glycerol, 6% SDS, 7.5% β-Mercaptoethanol, 0.75% Bromphenol blue in 200 nM Tris-HCL [pH 6.8]), which were subsequently heated at 95 °C for 5 min and spun down. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE healthcare life sciences). The WB membranes were blocked with 5% non-fat dry milk-TBST (10 mM Tris-HCL [pH 8.0], 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing 4 times for 5 min with TBST, membranes were incubated with secondary antibodies for 1 h at room temperature. The protein bands were detected using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) or Luminata Forte Western HRP substrate (Millipore, Billerica, MA) and visualized by exposure to X-ray film (GE healthcare life sciences). The following antibodies were used: rabbit monoclonal antibody to GAPDH (Cat Nr. 2118, Cell Signaling Technology), rabbit monoclonal antibody to p21 (Cat Nr. 2947s, Cell Signaling Technology), mouse monoclonal antibody to p53 (Cat Nr. sc-126, Santa Cruz Biotechnology), rabbit monoclonal antibody to acetyl-p53 (Lys382) (Cat Nr. 2525, Cell Signaling Technology), rabbit polyclonal antibody to NAMPT (Cat Nr. PAB17046, Abnova).

Whole-cell lysates were obtained by lysing equal numbers of cells with 3× laemmli buffer (30% glycerol, 6% SDS, 7.5% β-Mercaptoethanol, 0.75% Bromphenol blue in 200 nM Tris-HCL [pH 6.8]), which were subsequently heated at 95 °C for 5 min and spun down. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE healthcare life sciences). The WB membranes were blocked with 5% non-fat dry milk-TBST (10 mM Tris-HCL [pH 8.0], 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C. After washing 4 times for 5 min with TBST, membranes were incubated with secondary antibodies for 1 h at room temperature. The protein bands were detected using Pierce ECL Western Blotting substrate (Thermo Fisher Scientific) or Luminata Forte Western HRP substrate (Millipore, Billerica, MA) and visualized by exposure to X-ray film (GE healthcare life sciences). The following antibodies were used: rabbit monoclonal antibody to GAPDH (Cat Nr. 2118, Cell Signaling Technology), rabbit monoclonal antibody to p21 (Cat Nr. 2947s, Cell Signaling Technology), mouse monoclonal antibody to p53 (Cat Nr. sc-126, Santa Cruz Biotechnology), rabbit monoclonal antibody to acetyl-p53 (Lys382) (Cat Nr. 2525, Cell Signaling Technology), rabbit polyclonal antibody to NAMPT (Cat Nr. PAB17046, Abnova).