G18 promotes internalization of NRP1 and associates with NRP1 in LAMP2-marked structures. (A) Cells were incubated in medium alone or with 16 μg/mL sG16 at 37°C for 15 and 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa 488–conjugated anti–mouse IgG Ab. Nuclei were stained with DAPI. (B) Cells were incubated with medium alone (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 546–conjugated streptavidin (to visualize biotin-G18) and with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1). (C) Cells were incubated with medium only (top panels) or 16 μg/mL biotin-G18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with Alexa Fluor 488–conjugated streptavidin (to visualize biotin-G18) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). (D) Cells were incubated with medium only (top panels) or 16 μg/mL sG18 (bottom panels) at 37°C for 60 minutes. After fixation and permeabilization, cells were stained with anti-NRP1 primary mAb followed by Alexa Fluor 488–conjugated anti–mouse IgG Ab (to visualize NRP1) and with anti-LAMP2 primary Ab followed by Alexa Fluor 594–conjugated anti–rabbit IgG Ab (to visualize LAMP2). Cells were examined by confocal microscopy. Images were imported into Adobe Photoshop 6.0 for processing. Scale bar, 20 μm.