FIGURE 5.

GMFG knockdown does not affect β1-integrin endocytosis. Human monocytes or THP-1 cells were transfected with control negative siRNA(Ctrl siRNA) or GMFG siRNA. Forty-eight h after transfection, cell surface β1-integrin was labeled with anti-β1-integrin antibody at 4 °C for 1 h, followed by incubation of cells at 37 °C for the indicated time periods to induce internalization. A, analysis of β1-integrin internalization in siRNA-transfected human monocytes or THP-1 cells by flow cytometry. Surface-labeled cells were stained with Alexa Fluor 488-conjugated secondary antibody at 4 °C, followed by quantification of surface-remaining β1-integrin. Results are shown as the percentage of the integrin surface expression observed in control negative siRNA-transfected cells before internalization (i.e. the 0-min time point was set at 100%). Data represent the mean ± S.D. (error bars) of three experiments. B and C, analysis of β1-integrin internalization in siRNA-transfected human monocytes (B) or THP-1 cells (C) by immunofluorescence. Cells with surface-labeled β1-integrin were incubated at 37 °C for the indicated time periods to induce internalization, followed by stripping of surface-bound antibody by acid washing. Cells were then fixed, permeabilized, and stained with Alexa Fluor 488-conjugated secondary antibody at 4 °C. Nuclear DNA was labeled with DAPI (blue). Scale bars, 100 μm.