Fig. 4. Overexpression of NRP/B induced differentiation and hypophosphorylation of p110^RB in Tet-on/off PC12 cells. (A) The expression of NRP/B in Tet-on/off PC12 cells. Stable NRP/B transfected PC12 Tet-on/off cells were cultured in the presence or absence of tetracycline (1 µg/ml) for 48 hours. An equal amount of protein (5x10⁶ cells) was loaded and the blot was probed with monoclonal anti-NRP/B antibody for western blot (WB) analysis. SC10 represents the NRP/B Tet-off inducible clone and GH11 the Tet-on inducible clone. Bands indicate p67 NRP/B. (B) Morphological alteration of Tet-on PC12 cells upon tetracycline stimulation. GH11 clones were grown in 12-well plates coated with poly-D-lysine. Cells were unstimulated or stimulated with tetracycline (1 µg/ml) for 2 days and observed under a light microscope. (C) NRP/B induced hypophosphorylation of p110^RB. GH11 PC12 clones were stimulated with tetracycline as above. 30 µg total cell lysates were separated by 7.5% SDS-PAGE. The blot was probed with anti-RB antibody. The slow migrated form of p110^RB indicates hyperphosphorylated p110^RB. Bars, 10 µm.