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Fig. 5. (A) Effect of NRP/B and its mutants on cell cycle progression in PC12 cells. Subconfluent cells were transfected with wild-type NRP/B and its mutants for 48 hours. Cells were then labeled with propidium iodide. Mock (pEGFP), NRP/B-GFP (wt) and mutant NRP/B-GFP transfected cells were sorted out from non-transfected cells by flow cytometric analysis. Control, non-transfected cells. The y-axis represents the percentage of the cell population at each cell cycle stage. Data are the means±s.d. of duplicate samples. (B) Inhibition of the NRP/B-p110^RB association by mutation of the BTB domain. NRP/B-GFP and its mutants (BTB mutant A and kelch mutants that include mutation in the residues W518A or V4146, D418K) were cotransfected with various pRBWtHA-SVE constructs into 293T cells. The NRP/B-p110^RB interaction was analyzed by immunoprecipitation with anti-HA antibody, followed by probing with anti-GFP antibody (upper panel). The membrane was reprobed with anti-HA antibody (middle panel). To determine the transfection efficiency of the NRP/B constructs, total cell lysates were immunoprecipitated with GFP antibody followed by probing with anti-GFP antibody (lower panel). BTB mtA, BTB mutant A.

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