Supplemental Figure S1E-H -
Fig. S1. Inhibition of cell-surface-bound 125I-labelled TGFβ1 internalization by inhibitors of clathrin-dependent endocytosis in Mv1Lu cells. Cells were pretreated with vehicle only or 5 mg/ml β-CD (A,B,G), 20 µM TFP (A), 20 µM thioridazine (A), 20 µM MDC (B), 25 µM nystatin (C), 20 µM colchicine (D), 40 µM monensin (E), 200 µM chloroquine (F), 0.45 mM sucrose (G) or 10 and 40 µM dynasore (H) at 37°C for 1 hour. The binding of 125I-labelled TGFβ to cells was then performed at 4°C for 2 hours in the presence and absence of a 100-fold excess of unlabeled TGFβ1 (to estimate nonspecifically and total internalized TGFβ1, respectively). After washing, cells were warmed up to 37°C for different time periods, as indicated. Internalized cell-surface-bound 125I-labelled TGFβ1 was analyzed as described in the text. Specifically internalized 125I-labelled TGFβ (cell-surface 125I-labelled TGFβ internalized) was estimated by subtracting nonspecifically internalized 125I-labelled TGFβ1 from total internalized 125I-labelled TGFβ1. The vehicle and inhibitors were present throughout 125I-labelled TGFβ1 binding and 125I-labelled TGFβ internalization experiments. Experiments were carried out in triplicate. Each group of experiments (A-H) was performed at different times using 125I-labelled TGFβ with various specific radioactivities. Asterisk (*) indicates results significantly lower than control; P<0.001. For further details of the methodology, see the Materials and Methods section in article main text.
