Role of Dyn and Rab proteins in D6 surface expression and constitutive internalization. (A) Representative FACS analysis of CHO-K1/D6 cells transiently transfected with constructs expressing the indicated pEGFP-tagged proteins. R1 and R2 gates refer to the viable pEGFPneg and pEGFPhigh cells, respectively, in which D6 expression was evaluated. Histograms represent flow cytometric profiles of D6 expression in gated cell population (light gray = R1 gate; dark gray = R2 gate). Dashed lines indicate isotype-matched control mAb. (B) Quantification of D6 membrane expression. Data were expressed as percentage of MFI of R2 over R1 gated population. (C,D) D6 internalization of the indicated CHO-K1/D6 pEGFP-tagged transfectants (□ indicates pEGFP-N1 cells; ■, Dyn-K44A; ♦, Rab5-S34N; ○, Rab4-Q67L; ●, Rab4-S22N; △, Rab11-Q70L; and ▲, Rab11-S25N). Results are means plus or minus SEM from at least 3 independent experiments performed. Asterisks indicate significant differences of Dyn/Rab-transfected versus pEGFP-N1–transfected cells (*P < .05; **P < .01). (E) Confocal images of CHO-K1/D6 cells transiently transfected with pEGFP/Dyn-K44A (green) treated for 30 minutes with Alexa Fluor647–conjugated Tf (light blue) and immunofluorescence stained for D6 (red). Top panel (−): untransfected cells; bottom panel (+): pEGFP/Dyn-K44A–transfected cells.
