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Fig. 8. Inhibitory effect of NRP/B siRNA on neurite outgrowth in PC12 cells. (A) PC12 cells were either untransfected (a,b) or transfected with NRP/B siRNA (c,d) or with GFP siRNA (e,f). After 48 hours, cells were untreated (-) or treated (+) with NGF (50 ng/ml) as indicated and analyzed for neurite outgrowth. (B) Quantitative analyses of neurite outgrowth by depletion of NRP/B using the NRP/B siRNA approach. NRP/B siRNA inhibited neurite outgrowth in NGF-induced PC12 cells. The percentage of cells with neurites was measured after 48 hours of NGF treatment. 300 cells were analyzed in three independent experiments for each condition. Data are the means±s.d. Significantly inhibited neurite outgrowth was observed with NRP/B siRNA and NGF compared to NGF-induced neurite outgrowth with control siRNA (^*P<0.05). (C) Untransfected PC12 cells (UN), control GFP siRNA PC12 or NRP/B siRNA PC12 cells were harvested 48 hours after transfection and total mRNA was prepared. Semi-quantitative RT-PCR was performed using specific primers for NRP/B, which generated a product of 885 bp in length. As controls, we used specific primers for GAPDH and Mayven. (D) Effects of NRP/B siRNA on p110^RB phosphorylation. PC12 cells were seeded on poly-D-lysine-coated plates and transfected with NRP/B siRNA or GFP siRNA as a control. After 24 hours of transfection, cells were treated with NGF (50 ng/ml) for 24 hours. Total cell lysates were prepared and 100 µg of the lysates were analyzed by 7.5% SDS-PAGE. The blots were probed with anti-RB, phospho-RB (pRB Ser795), VD2 antibodies (for NRP/B) or with CSK antibodies (an internal control), as indicated. Bars, 10 µm.

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