Dock7, Sec16a, and Vac14 interact with Nbeal2. (A-B) Immunoprecipitation (IP) of Dock7, Sec16a, and Vac14 from PBW-FTAP HEKs with total cell lysate (input) in panel A, IP results in panel B. PBW-FTAP was detected by anti-FLAG, and a rabbit isotype immunoglobulin G (IgG) was used as control. Immunoblot with specific antibodies against Dock7, Sec16a, and Vac14 (top blot in panel B). The other 3 blots in panel B show the specific binding of each of the cognate antibodies, and the results with the isotype control immunoglobulin (ct-IgG) are in the first lane of the 4 blots in panel B. (C-E) Similar IP assays as in panel A carried out with HEKs (C) or CHRFs (D-E). (F) PLA with human MKs showing the results for (1) αIIb and GPIX as negative control (top left), (2) GPIbα and GPIX as positive control (top middle), (3) Sec16a (top right); Vac14 (2 bottom left); and Dock7 (2 bottom right); each interactor was tested in combination with Nbeal2. Interactions between proteins are presented as orange dots, and cells were counterstained with Phalloidin (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue). For better illustration of the PLA signals, a ×20 magnification of the insets in Vac14 and Dock7 are also shown. (4) Quantitative analysis of the number of interactions per cell surface based on analysis of the PLA signals by Image Studio are presented in the bar graph. Comparisons were made against the negative control. Bars represent mean ± SEM. *P value < .05.