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Fig. 2. Tlr exhibits slow Sog processing kinetics compared with Tld. (A) Time course of Sog processing by Tlr and Tld. The amount of Tlr in the reactions was fivefold more than the amount of Tld, as estimated by quantitative fluorescence measurements of the anti-HA immunoreactivity of the equivalently C-tagged Tld-HA and Tlr-HA contained in the bracketed area. The Dpp (R&D Systems) concentration was 3 x10^–10 M. The processing reactions were incubated at 25°C. Aliquots were removed at the indicated times, and were analyzed by immunoblotting with anti-Myc A14 antibody. The processing products were quantified using Odyssey Infrared Imaging System. The graphs below indicate the quantitation of the indicated bands at each time point. (B) Time course processing of Sog with increasing amounts of Tlr protein as indicated (10- and 25-fold excess when compared with Tld). (C) Time course of Sog processing in the presence of 1 x10^–9 M Dpp and variable amounts of Tlr protein as indicated. (D) Time course assays in which the Tld and Tlr protease domains have been swapped. In Tld^astTlr the Tld astacin domain has been replaced with the Tlr astacin domain, while Tlr^astTld is the converse construct. (E) Sog co-immunoprecipitates with Tlr and catalytically inactive Tld variants but not with active Tld. Equivalent amounts of wild-type and modified Tld-HA and Tlr-HA proteins were mixed with Sog-Myc for 3 hours at room temperature followed by immunoprecipitation with the anti-HA 12CA5 antibody. Sog was detected with anti-Myc A14 antibody.

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