Fig. 3. Co-translational phosphorylation of H1rev-P_N. COS-1 cells transfected with H1-P_N or H1rev-P_N were incubated with (+) or without (–) 100 µg/ml cycloheximide for 2 h before and 40 min during labeling with [³⁵S]methionine or [³²P]phosphate. After saponin extraction, the membrane proteins were immunoprecipitated and analyzed by SDS–gel electrophoresis and fluorography. The positions of phosphorylated H1rev-P_N with N_cyt/C_exo and N_exo/C_cyt orientation are indicated by an asterisk and an arrowhead, respectively. To visualize the total H1rev-P_N in the transfected cells, western analysis was performed on a parallel sample (W). A non-specific band detected upon western analysis is indicated by a circle.