Anti-Fas induces down-regulation of β-catenin–associated transcription and redistribution of β-catenin to cytoskeleton.

(A) Jurkat cells were transfected with 10 μg pTOPFLASH or pFOPFLASH, incubated for 18 hours, and further incubated for the time indicated in the presence of anti-Fas antibody (100 ng/mL). Cells were then lysed, and luciferase activity was assayed as described in “Materials and methods.” (B) Immunoblot analysis. Jurkat cells were incubated with anti-Fas (100 ng/mL) for the indicated times and extracted in CSK buffer, and β-catenin was immunoprecipitated with anti–β-catenin antibody directed to amino acids 571 to 781. Immunoprecipitates and total cell lysates were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed either with antibody to β-catenin or actin. (C-F) Immunocytochemical analysis. β-Catenin distribution was analyzed in a standard preparation (C,D) and in a cytoskeletal preparation (E,F), in which soluble proteins were removed by incubation in 1% Triton X-100. Control (C,E) and 30-minute anti-Fas–treated (D,F) Jurkat cells were stained with anti–β-catenin antibody C571-781 followed by Cy3-conjugated goat antimouse antibody. (Magnification, ×630).