FIG 3.

MXRA8 internalization occurs independently of its transmembrane and cytoplasmic domains as shown by antibody labeling. (A and B) ΔMxra8 MEFs were complemented with WT MXRA8, MXRA8 lacking its cytoplasmic domain (ΔC-tail), or MXRA8 lacking both transmembrane and cytoplasmic domains but fused with rodent herpesvirus Peru (RHVP)-encoded or placental alkaline phosphatase (PLAP)-encoded GPI anchors. Cells were incubated with anti-MXRA8 antibody at 37°C for 30 min. After blocking of uninternalized anti-MXRA8 on the cell surface, the internalized MXRA8 and early endosome marker EEA1 were stained for confocal imaging. Orthogonal views (A) and spot-based colocalization (B) were processed using Imaris software. The percentage of colocalized EEA1 (B, left column) with MXRA8 of the total number of EEA1 and the percentage of colocalized MXRA8 (B, right column) with EEA1 of the total number of MXRA8 were calculated. One representative image from at least three independent experiments is shown; scale bar, 5 μm. (C) Internalized MXRA8 and the lysosomal marker LAMP1 were stained for confocal imaging. Spot-based colocalization was analyzed using Imaris software. The percentage of colocalized LAMP1 (C, left column) with MXRA8 of the total number of LAMP1 and the percentage of colocalized MXRA8 (C, right column) with LAMP1 of the total number of MXRA8 were calculated. (D) Cell surface expression of MXRA8 in complemented cells that express different forms by flow cytometry. A representative image of at least two independent experiments is shown. (E) CHIKV infection as measured by E2 antigen expression in complemented MEFs by flow cytometry. Data are an average of two independent experiments performed in triplicate (mean ± SD).