Mouse anti cdc25a

Purified FLAG-Cdc25A, GFP-ARD1, His-HDAC11, and corresponding concentrations of BSA (as control) were applied directly onto nitrocellulose membranes (at the concentrations indicated in figures). The samples were dried for 15 min at 4°C. The membranes were blocked for 1.5 h at 4°C with 5% nonfat dry milk in TBST, followed by incubation with 5% milk solution containing purified FLAG-Cdc25A, GFP-ARD1, or His-HDAC11 for 3 h at 4°C. Membranes were washed 3 times, for 10 min each, with TBST, and were subjected to immunodetection with mouse anti-Cdc25A (Santa Cruz Biotechnology), mouse anti-GFP (Jackson ImmunoResearch), mouse anti-FLAG (Agilent Technologies), or mouse anti-His (Oncogene) antibodies for 1 h at 4°C (see figure legends for specifics of each experiment). The incubation with the appropriate HRP-linked secondary antibodies (Santa Cruz Biotechnology) was for 1 h at room temperature. Signal was detected using ECL Plus and visualized by autoradiography.