gH/gL triplex formation with GP129, GP131, GP133 or gO.
GP129, GP131, GP133 and gO were evaluated for an ability to form triplex complexes with gH and gL. Transient expression of epithelial cells with gHGFP, gLmCherry, GP129myc, GP131HA and GP133FLAG was as described in materials and methods. Evaluation of triplex formation was by cellular colocalization or by GFP trap immunoprecipitation assay. A-E. gHGFP/gLmCherry/GP129myc triplex formation. Panels A-D, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (D). Western blot of triplex immunoprecipitation (E). Lanes 1, 4 and 7 (total cell lysate). Lanes 3, 6 and 9 (IP). Lanes 2, 5 and 8 (flow through wash). F-J. gHGFP/gLmCherry/GP131HA triplex formation. Panels F-H, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (I). Western blot of triplex immunoprecipitation (J) as described for E except lanes 7–9 GP131HA western. K-O. gHGFP/gLmCherry/GP133FLAG triplex formation. Panels K-M, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (N). Western blot of triplex immunoprecipitation (O) as described for E except lanes 7–9 GP133FLAG western. P-T. gHGFP/gLmCherry/gOFLAG triplex formation. Panels P-R, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (S). Western blot of triplex immunoprecipitation (T) as described for E except lanes 7–9 gOdelFLAG. Cellular co-localization merged figures (D, K, N and S) include DAPI co-stained cells. GP129myc, GP131HA, GP133FLAG and gOdelFLAG detected by primary anti-epitope antibody and secondary anti-mouseIgG-Cy5 (immunofluorescence) and anti-mouseIgG-HRP (western blot). Both gHGFP and gLmCherry were detected by fluorescence (cell localization) and specific epitope antibody (western). Panels E, J, O and P western blots. Lanes: 1, 4 and 7 total cell lysate; 2, 5 and 8 wash flow through; 3, 6 and 9 immunoprecipitation.
