Peroxidase conjugated goat anti rabbit igg

Cells in primary culture (27 × 10⁴ cells/well in 6-well culture plates) were allowed to grow until nearly confluence and then treated with IL-1β (10 ng/ml) or IL-1β+CM for different times. Cells were lysed with buffer (1% Triton X-100, 1% deoxycholic acid, 20 mM NaCl and 25 mM Tris, pH 7.4) and centrifuged at 4°C for 10 minutes at 10000 xg. Protein content was determined by the DC Bio-Rad Laboratories protein reagent (Bio-Rad, Madrid, Spain). Proteins (25 μg) in supernatants were separated by SDS/PAGE (12.5% gel) and transferred on to PVDF membranes. Membranes were blocked with 3% BSA and incubated with specific polyclonal antibodies against p53 (Novus Biologicals, Littleton, CO, USA), acetyl-p53 (ChemiconMillipore Iberica, Madrid, Spain), P-p38 (Promega Corp., Madison, WI, USA), p38, JNK1/2, P-JNK1/2, ERK1/2, P-ERK1/2 (Cell Signalling Technology Inc.) overnight at 4°C. Membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG (Dako, Copenhagen, Denmark); for p53 we used the secondary antibody anti-mouse IgG (Fc-specific)-peroxidase (Sigma-Aldrich). Finally, membranes with the immunoreactive bands were visualized by ECL® (enhanced chemiluminescence; GE Healthcare, Barcelona, Spain) using an AutoChemi image analyser (UVPInc., Upland, CA, USA).