Fig. 1. (A) 8ß1 adhesion to LAP-TGFß1. Mock transfected or 8-transfected cells (50,000/well) were allowed to attach to wells precoated with 5 µg/ml of recombinant LAP-TGFß1. In some cases, cells were incubated with v integrin antibody (L230) or ß1 integrin antibody (5D1) prior to adhesion assay. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD₅₉₅. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (B) Adhesion of SW4808 or SW480ß6-transfected cells to increasing concentrations of LAP-TGFß1 with or without 1mM Mn²⁺. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD₅₉₅. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (C) Adhesion of 8 or ß6-transfected cells to fibronectin. AtT208, SW4808 or SW480ß6 cells were allowed to attach to wells precoated with the indicated concentration of fibronectin. After 1 hour, non-adherent cells were removed by brief centrifugation and adherent cells were fixed and stained with formaldehyde/crystal violet. Adherent cells were quantitated by measuring absorbance of wells at OD₅₉₅. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well. (D) Mutation of RGD site in LAP TGFß1 eliminates 8ß1 adhesion. 8-transfected cells were allowed to attach to wells coated with 5 µg/ml of authentic recombinant LAP-TGFß1 (RGD-LAP-TGFß1) or recombinant LAP-TGFß1 containing a single glutamic acid for aspartic acid substitution mutation in the RGD site (RGE-LAP-TGFß1) for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of BSA-coated well. (E) Adhesion of AtT208 cells to LAP-TGFß3 is similar to adhesion to LAP-TGFß1. AtT20 or AtT208 cells were allowed to adhere to increasing concentrations of recombinant LAP-TGFß1 or LAP-TGFß3 for 1 hour; adhesion was then assessed by absorbance. Data are reported as the average of triplicate wells±s.e., minus the mean absorbance of the BSA-coated well.