Figure 6.

ER retention of the H₁R^R allele requires the L-M-S haplotype. (a) HEK293T cells were transfected with empty control, single HA-H₁R^S mutants or HA-H₁R^R plasmids. Cells were collected 16–24 h later without trypisinization, stained with anti-HA mAb and analyzed by flow cytometry. Cells transfected with HA-H₁R^S are shown as positive controls in the far-right panel. The thin line represents cells transfected with empty pEGZ whereas the thick line represents cells transfected with HA-H₁R^S and the filled area represents cells transfected with HA-H₁R^R. (b) HEK293T cells were analyzed as in (a) and the mean florescence intensity of anti-HA on H₁R^S positive cells was determined. The data presented is the average of triplicate transfections. (c) HEK293T cells were co-transfected with pdsRed plasmid that express ER targeted dsRed protein (red) and HA-H₁R^S, mutants of HA-H₁R^S or HA-H₁R^R. 24h later, cells were fixed, permeabilized, stained with anti-HA mAb (green) and the co-localization of HA-H₁R with dsRed (red) was visualized by confocal microscopy. Yellow color represents the co-localization of red and green colors. (d) Quantification of HA-H₁R co-localization with dsRed protein. Using Zeiss LSM 510 META Confocal imaging software the number of pixels expressing both the colors was determined in a number of cells (n ≥ 16) and the data is presented as the average of number of pixels that co-express dsRed and HA-H₁R. Error bars indicate SEM.