Fig. 3. NRP/B interaction with p110^RB is mediated via the BTB domain. (A) The association of the NRP/B-BTB domain with p110^RB. NRP/B-GFP constructs were cotransfected with pRBWtHA-SVE constructs into 293T cells. Total cell lysates were immunoprecipitated with anti-HA monoclonal antibody. NRP/B association with pRB was observed by probing with anti-GFP polyclonal antibody. The membrane was reprobed with anti-HA antibody (bottom panel). Lane 1, IgG control; lane 2, full-length NRP/B-GFP; lane 3, NRP/B-BTB-GFP; lane 4, NRP/B-kelch-GFP. (B) Specific association of the NRP/B-BTB domain to the B pocket of p110^RB. A schematic presentation of the pRB-HA-SVE constructs (upper panel). NRP/B-GFP constructs (full-length, NRP/B-BTB, and NRP/B-kelch) were cotransfected with wild-type p110^RB or with the p110^RB deletion mutant (del9.1 in A pocket) into 293T cells. Total cell lysates (500 µg) in GFP buffer were immunoprecipitated with anti-HA antibody and blotted with anti-GFP monoclonal antibody or with anti-HA antibody (lower panels). pRB, p110^RB.