Fig. 1. Characterization of the anti-phospho-c-Jun antiserum. a, GST-c-Jun(1–223) (lanes 1, 2) or GST-c-Jun(1–223, A63/73) (lanes 3, 4) were (+) or were not (−) phosphorylated by recombinant JNK2 in the presence of [32P]ATP.(Top to bottom) first panel, Autoradiogram of32P-labeled proteins exposed either overnight (o/n) or (second panel) exposed only for 2 min. Third panel, The same blot was probed with affinity-purified phospho-c-Jun (α-P-cJun) antibody. Fourth panel, The blot was stripped and reprobed with the c-Jun antiserum (α-cJun).b, Samples (0.1 μg, lanes 2, 3; 1.0 μg, lanes 1, 4) of recombinant full-length c-Jun (Deng and Karin, 1992) were not (lanes 1, 2) or were phosphorylated (lanes 3, 4) with recombinant JNK2 in the presence of [32P]ATP (lanes 3, 4). (Top to bottom) first panel, Autoradiogram of the 32P-labeled proteins. Second panel, Immunoblotting with the phospho-c-Jun antiserum (α-P-cJun). Third panel, The blot was stripped and treated with buffer containing heat-inactivated calf intestinal phosphatase (
) or (fourth panel) native CIP (40 U/ml).Fifth panel, After final stripping, the blot was reprobed with the c-Jun antiserum (α-cJun).c, Immunodetection of phosphorylated c-Jun in immortalized 3T3 fibroblasts derived from wild-type (lanes 1, 2) or c-jun−/− mouse embryos (lanes 3, 4) (Hilberg et al., 1993) or c-jun−/−cells stably transfected with a human c-jun expression vector (lanes 5, 6), which were (+) or were not (−) UV-irradiated. The membrane was probed with the phospho-c-Jun antiserum (α-P-cJun) or c-Jun antibody (α-cJun). d, Detection of phosphorylated c-Jun in nuclear cortical extracts from untreated rats (lane 1) or after ischemia with 24 hr reperfusion (lane 2) by immunoblotting with the anti-phospho-c-Jun or anti-c-Jun antiserum.