Fig 8.

Clathrin-coated pits are required for LRP-1-mediated endocytosis of CD44 in FTC-133 cells. FTC-133 cells were transfected with nontargeting siRNA sequences (siCTRL) or siRNA targeting LRP-1 (siLRP-1) for 48 h (A) or treated with 500 nM RAP for 1 h (B and C), followed by treatment with 5 mM MβCD for 45 min (B) or 400 mM sucrose for 1 h (C). Cells were then transferred to ice, and cell surface proteins were biotinylated. Then, tumor cells were transferred to 37°C to permit endocytosis for 30 min in complete medium (with or without RAP, MβCD, or sucrose). Cells were then placed on ice, washed, and incubated with pronase (1 mg/ml) to remove surface-bound proteins. Biotinylated proteins were recovered by avidin protein immobilized to agarose beads, subjected to SDS-PAGE, and revealed by immunoblotting for CD44 and stand for the endocytosis fraction, as previously reported (59). Binding of CD44 to the cell surface and the efficiency of pronase were controlled in panel A (left frame, lanes 1 and 2, respectively). Histograms (right frames) represent the quantification of internalized CD44. Results are expressed as percentages compared to results for control cells (A) or wild-type nontreated cells (B and C), which serve as a reference. n.s., not significant; *∗, P < 0.01.