Figure 2
Cf expressed TMD0 is correctly folded. (A,B) Interaction of coreTAPL with TMD0. HEK 293 T cells were transiently transfected with coreTAPL and TMD0-myc (A) or cf-TMD0-myc (B). After solubilization with 1% digitonin (input), co-immunoprecipitation with myc-tag specific antibody (IP myc) or isotope control (mock) was performed and analyzed by immunoblotting. Input represents 1/25 aliquot of the volume used for immunoprecipitation. Original, uncropped immunoblots are shown in supplementary information. (C) To probe correct folding of cf expressed TMD0, cf-TMD0-His was reconstituted into proteoliposomes alone or together with coreTAPLmV or TmrAB (input). After solubilization with 2% digitonin, coreTAPLmV or TmrAB were pulled-down with ATP-agarose and analyzed by immunoblotting using a His-tag antibody. Input represents 1/20 aliquot of the volume used for pull-down. Original, uncropped immunoblots are shown in supplementary information. (D) To confirm functional reconstitution, proteoliposomes containing only cf-TMD0-His or cf-TMD0-His and coreTAPLmV or TmrAB were incubated in the presence (filled bars) or absence (open bar) of ATP (3 mM) with peptide (3 µM) for 15 min at 37 °C. Transport assay was performed in triplicates and normalized, error bars indicate SD.
