Fig. 9. In vivo CHL1 phosphorylation status under different nitrogen conditions. (A) Specificity of anti-P-T101 antibodies for phosphorylated CHL1 protein. Membrane proteins were isolated from the root tissue of wild-type (W) or chl1-5 (C) plants and treated with (+) or without (–) CIP for 60 min, then 20 µg of protein was loaded in each lane; after electrophoresis and transfer, the blots were probed with anti-CHL1 antibodies (left) or anti-P-T101 antibodies (right). (B) Regulation of CHL1 phosphorylation by nitrogen conditions. Ten-day-old wild-type plants were shifted to 150 µM NH₄NO₃ (Low N) or 12.5 mM NH₄NO₃ (High N) medium for 2.5 h, then membrane proteins were isolated. The upper panel shows western blots probed with anti-CHL1 antibodies and the lower panel those probed with anti-P-T101 antibodies. Membrane protein from the chl1-5 mutant was loaded in the right-hand lane as a negative control.