A. Detection of mouse p19ARF by immunoblotting. Samples: Whole cell lysate (25 µg) from NIH3T3 cells that have deleted the Arf gene (lane 1) and wild type mouse embryo fibroblasts (MEFs) at passage 6, which express p19ARF (lane 2). Primary antibody: Anti-p19ARF (54-75) Rat mAb (12-A1-1) (Cat. No. CB1013) (1 µg/ml). Detection: chemiluminescence. B. Detection of mouse p19ARF by immunoprecipitation. Samples: Whole cell lysates (250 µg) from NIH3T3 cells that have deleted the Arf gene (lane 5) and NIH3T3 cells expressing p19ARF with a HA-tag (lanes 3,4). Antibodies for IP: Normal rat IgG (negative control; lane 3) and Anti-p19ARF (54-75) Rat mAb (12-A1-1) (Cat. No. CB1013). Conditions for immunoblotting: Samples: Whole cell lysate (25 µg, equivalent to 10% of above immunoprecipitations) from NIH3T3 cells that have deleted the Arf gene (lane 1), NIH3T3 cells expressing p19ARF with a HA-tag (lane 2), and immunoprecipitated proteins as indicated above. Primary antibody: Anti-HA. Detection: chemiluminescence. C. Detection of mouse p19ARF by immunocytochemistry. Samples: Wild type mouse embryo fibroblasts (MEF) at passage 5 (panels 1,2) and NIH3T3 cells, which have deleted the Arf gene (panels 3,4), fixed in methanol/acetone. Primary antibody: Anti-p19ARF (54-75) Rat mAb (12-A1-1) (Cat. No. CB1013). Detection: Anti-Rat IgG secondary antibody, fluorescent conjugate (panels 1,3) and counterstained with Hoechst dye (panels 2,4).
Detection of mouse p19ARF by immunoblotting. Samples: Whole cell lysate (30 µg) from MEF (lanes 1,3) and NIH3T3 (lanes 2,4) cells. Primary antibody: Anti-p19ARF (54-75) Rat mAb (12-A1-1) (Cat. No. CB1013) (3 µg/ml) (lanes 1,2) or Anti-p19ARF (54-75) Rat mAb (5-C3-1) (Cat. No. CB1012) (3 µg/ml) (lanes 3,4). Detection: chemiluminescence.
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