Fig 1: HEF with mutations in putative contact amino acids is retarded in intracellular transport.A, HEK-293T cells expressing the indicated HEF proteins were lysed after transfection, and an aliquot was subjected to SDS-PAGE and Western blotting with anti-HEF antiserum and anti-actin antibodies as loading control. B, quantification of surface exposure of HEF mutants by flow cytometry. HEK-293T cells were fixed at 48 h after transfection and either directly stained with anti-HEF antiserum (surface expression) or permeabilized prior to staining (total expression). The mean fluorescence intensity (MFI) from at least 10,000 cells of three different transfections was determined by flow cytometry. The results were normalized against the surface expression of WT HEF for each transfection, and the relative surface expression is plotted against the total expression for each protein. The asterisks indicate statistically significant differences (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001) between WT and the mutants. One-way ANOVA followed by Tukey’s multiple comparison test was applied for statistical analysis of three independent experiments. C, localization of HEF in transfected cells. CHO-K1 cells were transfected with WT (upper panel), or with F210Y213 (2S, second from above), or with Y101L102Y103 (3S, third panel), or with F210Y213 + Y101L102Y103 (5S, forth panel) and R322 + E350 (lower panel). HEF was stained with polyclonal anti-serum against the HEF followed by secondary anti-rabbit antibodies coupled to Alexa Fluor-488 fluor, ER with ER staining Kit-Red Fluorescence-Cytopainter form Abcam and nuclei with DAPI. Scale bar: 5 μm. D, colocalization of HEF with ER from at least 40 cells from three different transfections was quantified with the Pearson’s correlation coefficient method using the JACoP plugin of the ImageJ software. The asterisks indicate statistically significant differences (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001) between WT and the mutants. One-way ANOVA followed by Tukey’s multiple comparison test was applied for statistical analysis. CHO-K1, Chinese hamster ovary cells; ER, endoplasmic reticulum; HEF, hemagglutinin-esterase-fusion glycoprotein; HEK-293T, human embryonic kidney-293T cells.