PAL-E recognizes PV-1, which can form complexes with NRP-1. (A) HEK-EBNA cells mock transfected (HEK-mock), transfected with PV-1 (HEK-PV-1) or NRP-1 (HEK-NRP-1), were stained with the indicated antibodies and analyzed by flow cytometry. In the histograms, the x-axis represents the fluorescence intensity; and the y-axis, the number of cells. (B) Immunoblots of HEK-PV-1 transfectants and tonsil lysate. The indicated antibodies followed by HRP-conjugated anti–mouse IgG or anti–sheep IgG were used for chemiluminescence detection (neg. contr. 1 and neg. contr. 2 indicate controls for anti–PV-1 and PAL-E, respectively; and neg. contr. 3, control for anti–NRP-1). Gels were run under nonreducing conditions, allowing the maintenance of PV-1 homodimers, resulting in PV-1 bands of approximately 110 to 130 kDa and NRP-1 bands of approximately 130 to 140 kDa. (C) Coimmunoprecipitations with anti–PV-1 and anti–NRP-1 antibodies. The precipitating and detecting antibodies are indicated (neg.-1 or neg. contr.-1 indicate the negative control antibody for anti–PV-1; and neg.-2 or neg. contr.-2, the negative control antibody for anti–NRP-1, respectively). Gels were run under reducing conditions, thus showing monomeric PV-1 bands of approximately 55 to 65 kDa and NRP-1 bands of approximately 130 kDa. Arrows point to the coprecipitated proteins in lane 2 and lane 5. (D) HEK-EBNA cells transiently cotransfected with PV-1 and NRP-1 were used to confirm the results of the coimmunoprecipitation from fresh tonsil lysate. Gels were run under reducing conditions, thus showing monomeric PV-1 bands of approximately 55 to 65 kDa and NRP-1 bands of approximately 130 kDa. The arrow indicates the coprecipitated protein in lane 1. Bands in the range of 50 kDa stem from the heavy chain of the immunoglobulins. The experiments were performed 3 times (A) and twice (B-D) with comparable results.