Fig. 2

Arf6-KD decreased CD147 recycling to the cell surface. Arf6-perturbed liver cancer cells were incubated with H18Ab for uptake at 37 °C. After washing surface-residual Ab with PBS/1 M NaCl, cells were chased for 30 min and the recycled CD147-H18Ab complex on the cell surface was stained with anti-mouse AF488 and imaged under a confocal microscope. In parallel, chased cells were further washed with PBS/1 M NaCl to remove the surface-recycled portion, then fixed, permeabilized, and the intracellular non-recycled complex (cytosol) was stained and confocal imaged. a Representative observations are shown. Scale bar: 20 um. b, c Box-and-whiskers plots depict the surface (recycled) and cytosol (non-recycled) CD147 pool in cell populations. The recycled or non-recycled CD147 pool in untreated cells (WT) was set as 100%. Significant differences compared with NC-KD cells are shown. Western blot determined the biontin-CD147 level after cell surface biotinylation, avidin binding, and DTT elution. Representative blot results from three independent experiments are shown (d, e, f), and corresponding quantitative scans of CD147 blots were analyzed (g, h, i). The cell-surface biotin-CD147 level in untreated cells (WT) was set as 100%. Significant differences compared with NC-KD cells are shown. n = 3. ** P < 0.01