Fig. 5. Blocking of β1 integrin inhibits fibronectin endocytosis. (A-C) FN-null myofibroblasts were incubated with 25 µg/ml β1 antibodies (Ha2/5) or isotype control antibodies at 4°C for 30 minutes. 10 µg/ml TR-fibronectin (A,B) or AF488-fibronectin (C) was then added to the medium and cells were processed for fibronectin endocytosis pulse-chase assays. After 2 hours of chase, cells were either fixed for imaging assay (A, β1 inhibition; B, isotype control), or processed for flow cytometry to quantify endocytosed fibronectin (C). The numbers over the peaks in C are the MFI of internalized AF 488-fibronectin. (D-F) Smooth muscle cells were seeded in serum-free medium on vitronectin-coated dishes. Cells were allowed to adhere for 3 hours and then processed for integrin-blocking assay as described above for FN-null myofibroblasts. (D) 25 µg/ml β1 inhibitory antibodies (Ha2/5); (E) isotype control; blue: DAPI. (F) Quantification of endocytosed fibronectin in smooth muscle cells by flow cytometry. The numbers over the peaks in F are the MFI of internalized AF 488-fibronectin. Scale bars: 10 µm.