Evaluation of the binding and endocytosis of3H-FA and FA-labeled bovine serum albumin by cultured cells lacking (A549) or expressing (A549/FR-β) the β isoform of the FR. (A) A549 cells, a human lung carcinoma cell line expressing no detectable FR, were transfected with the β isoform of FR (A549/β-FR) and examined by flow cytometry for expression of the β isoform, as described in Materials and Methods. The filled peaks correspond to cells treated only with preimmune serum, while the open peaks show cells labeled with anti-FR IgG. (B) Cells were incubated with 100 nmol/L 3H-FA for 2 hours at 37°C and then washed three times in PBS, pH 7.4, to remove unbound ligand (□) or three times in sodium acetate buffer, pH 3.5, to strip all externally bound ligand from the cells (▨). (C) Binding and endocytosis of 125I-labeled serum albumin (100 nmol/L) was conducted similarly, except to distinguish specific from nonspecific binding, uptake was also evaluated in the presence of a 10,000 times molar excess of free FA to competitively block all folate-specific sites (▩).
