Secondary antibody conjugated to horseradish peroxidase

Cells were lysed on ice in buffer containing 10 mmol/L HEPES (pH 7.9), 10 mmol/L potassium chloride, 0.1 mmol/L EDTA, 0.2 mmol/L ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and 0.5% Nonidet P40 supplemented with 1 mmol/L dithiothreitol, 10 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L Na3VO4, and 1 mmol/L sodium fluoride. Lysates were clarified by centrifugation and separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Blots were incubated with antibodies against p-p38 (1:1000, #4511S; Cell Signalling Technology, Danvers, MA), p38 (#sc-7972), phosphorylated extracellular signal–regulated kinase-1/2 (#sc-7383), phosphorylated c-Jun N-terminal kinase (#sc-6254) (1:500; all from Santa Cruz Biotechnology), and β-actin antibody (1:5000, #A544; Sigma), followed by a secondary antibody conjugated to horseradish peroxidase (1:20,000; Dako, Santa Clara, CA).

Protein extracts were prepared and run as previously described.^{22 (link)} Blots were incubated with Smad7 (1 μg/ml, R&D Systems), p-CDK2 (Thr-14/Tyr-15) (sc-28435-R), CDK2 (sc-6248), CDC25A (sc-7389), CDC25B (sc-5619), CDC25C (sc-13138), eIF2-α antibodies (1 : 500 final dilution, all from Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-eIF2α Ser51 (1 : 1000 final dilution, Cell Signaling Technology, Danvers, MA, USA), followed by a secondary antibody conjugated to horseradish peroxidase (Dako, Milan, Italy). After analysis, each blot was stripped and incubated with a mouse–anti-human monoclonal β-actin antibody (1 : 5000 final dilution) to ascertain equivalent loading of the lanes. Computer-assisted scanning densitometry (Total Lab, AB.EL Science-Ware Srl, Rome, Italy) was used to analyze the intensity of the immunoreactive bands.