GPS-associated missense variants abrogate NBEAL2-SEC22B binding, and NBEAL2 is required for SEC22B–P-selectin interaction. (A) Two GPS-associated missense variants E1833K and R1839C are located within the putative SEC22B binding region of NBEAL2 (aa 1798-1903). (B) Transfected HEK293 cells coexpressing FLAG-SEC22B and either HA-tagged WT NBEAL2 (lanes 1-3), NBEAL2 (E1833K) (lanes 4-6), or NBEAL2 (R1839C) (lanes 7-9) were used for IP experiments and analyzed via immunoblots. Although all HA-tagged constructs were expressed, WT NBEAL2, but not the 2 NBEAL2 variants was able to coimmunoprecipitate SEC22B. (C) Similar experiments performed with HEK293 cells coexpressing GFP-tagged P-selectin instead of FLAG-SEC22B showed that WT NBEAL2 and both NBEAL2 variants were able to coimmunoprecipitate P-selectin, indicating a specific effect of GPS-associated variants on SEC22B binding. (D) Native NBEAL2 and SEC22B were immunoprecipitated using specific antibodies in WT, NBEAL2-KO, and SEC22B-KO imMKCL cells. NBEAL2, P-selectin, and SEC22B can be coimmunoprecipitated together in WT cells (WT, left panel). However, SEC22B was unable to coimmunoprecipitate P-selectin in the absence of NBEAL2 (NBEAL2-KO, middle panel), whereas NBEAL2 could coimmunoprecipitate P-selectin in the absence of SEC22B (SEC22B-KO, right panel). These results suggest that NBEAL2 mediates the interaction between SEC22B and P-selectin.