Figure 5
From: Caspase cleavage of the Golgi stacking factor GRASP65 is required for Fas/CD95-mediated apoptosis
GRASP65 and Fas/CD95 do not directly interact at the Golgi apparatus. (a) GST-tail pull-down assay. Comparison of the C-terminal sequence of GM130 – a protein known to interact with GRASP65 – and the fusion proteins used in the assay (above). Example tail-binding assay showing strong binding of GRASP65 to the C-terminus of GM130, but only weak binding to both wild-type and mutant tail fusions of Fas/CD95 (below). (b and c) Immunoprecipitation of endogenous Fas/CD95 (b) and GRASP65 (c) and immunoblotting for the respective candidate binding partner. (d and e) GFP–Trap immunoprecipitations of HeLa cells transiently expressing wild-type or tail mutant Fas–GFP (d), and GFP–Trap immunoprecipitations of HeLa cells stably expressing GRASP65–GFP (e) immunoblotted using the antibodies shown. <, Endogenous Fas/CD95 or GRASP65; <<, GFP-tagged Fas/CD95 or GRASP65
