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Rabbit anti-mouse phospho-p38 MAPK, phos-pho-JNK, phospho-ERK and Rabbit anti-mouse [beta]-actin antibody was purchased from Cell Signaling Technology (Boston, MA, USA), and horseradish peroxidase-conjugated goat anti-rabbit antibody, bicinchoninic acid (BCA) protein assay kit and enhancer chemiluminescent (ECL) reagents were obtained from Pierce Biotechnology (Rockford, IL).
Rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate 1 (FITC; 1:100 in 1% BSA in PBS; Santa Cruz Biotechnology, Santa Cruz, Calif) was used as a secondary antibody.
Finally, the difference between goat anti-mouse and rabbit anti-mouse secondary horseradish peroxidase-labeled antibodies was investigated.
After washing with T-PBS, strips were incubated for 30 min at room temperature with a 1:1000 dilution of rabbit anti-mouse immunoglobulin or a 1: 2000 dilution of rabbit anti-human IgG F(ab')Z conjugated with horseradish peroxidase (DAKO).
Chicken antibody-based assays have been tested using a rabbit anti-mouse antibody (HAMA surrogate) and with sera from HAMA-positive patients (treated with monoclonal antibody 17-1A).