Fig. 2. (A) Representative immunoblot analyses of cell-cycle-regulatory proteins. NGF-differentiated mock and p25 cells were treated (+) or not (–) with tetracycline for 24 hours and lysates were analysed using antibodies against p21^CIP1 (p21), p27^KIP1 (p27), cyclin A, cyclin B1 and p34^Cdc2. Equal amounts of proteins were loaded, and visualized with an antibody against neuron-specific -enolase (NSE). (B) Analysis of Rb phosphorylation in a time-course experiment of p25 expression. Lysates from NGF-differentiated mock and p25 cells treated (+) or not (–) with tetracycline for 6 hours, 12 hours and 24 hours were first immunolabelled with antibodies against Ser807/811-phosphorylated and Ser795-phosphorylated Rb. After stripping, phosphorylation-independent antibody (IF-8) was used to visualize the amount of loaded Rb (Rb). Concomitant with this, p25 expression was analysed during the kinetics by antibody against the p35 C-terminus.