Fig. 1. Biochemical characterization of the Tlr protein. (A) Western blotting analysis of the Tld-HA, Tlr-HA proteins and various modified forms. Shown are supernatants (s) and soluble cell fraction (c) from transfected S2 cells probed with anti-HA 12CA5 antibody. The bracket on the left indicates position of unprocessed and partially processed forms. The amount of secreted Tld is always low and the band is often smeared due to glycosylation (Marqués et al., 1997). (B) Comparison of Sog processing by Tld and Tlr, in the absence or presence of Dpp and Tsg. The indicated combinations of proteins were incubated for 16 hours at 25°C and Sog fragments were visualized with anti-Myc A14 antibody. Under these conditions, processing is incomplete, enabling the different fragments to be seen. The molecular weight of full-length Sog is 120 kDa and the C-terminally Myc tagged products shown are of 110 kDa, 50 kDa and 25 kDa. The 25 kDa band in the Tld lane is not visible with this level of exposure. (C) Schematic representation of the Tld and Tlr cleavage sites. The position of cleavage site II is indicated but this site is very weak for Tld (Shimmi et al., 2003) and undetectable for Tlr. Sog processing by Tlr requires an active, enzymatically intact astacin domain. (D) Equivalent amounts of wild-type and modified Tlr proteins were tested for their Sog processing activity by incubation for 40 hours at 25°C in the combinations indicated. Processing at site I only (full-length Sog versus the 110 kDa fragment) is shown. pmTlr, processed mutant Tlr; cdTlr, catalytically dead Tlr.