Fuji fla 3000 phosphor imager

In vitro protein synthesis was performed as previously described [12 (link),18 (link)]. Briefly, rabbit reticulocyte lysate (Promega) was supplemented with 19 amino acid mix and [³⁵S] methionine/cysteine in the presence of canine pancreatic microsomes (gift from Professor Bernhard Dobberstein, University of Heidelberg). The mix was incubated at 30°C for 15 min, aurintricarboxylic acid added to 0.1 mM to stop further translation initiation, and samples incubated for an additional 15 min. Membranes were isolated by centrifugation through a high salt cushion (750 mM sucrose, 500 mM KOAc, 5 mM Mg(OAc)₂, 50 mM HEPES-KOH pH 7.9) at 100000×g for 10 min. Pelleted microsomes were resuspended in 0.1 M Na₂CO₃, pH 11.5 and incubated for 15 min on ice to enrich for integral membrane proteins [22 (link)], the membrane fraction re-isolated by centrifugation and resuspended in Laemmli sample buffer. To assess protein N-glycosylation, microsomes were directly resuspended in Laemmli sample buffer and treated with Endoglycosidase H (EndoH, New England Biolabs, Ipswich, MA, U.S.A.) or buffer control at 37°C for 4 h. Proteins were separated using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), the gels were then fixed and dried then exposed on phosphor-imager plates which were read using a Fuji FLA-3000 Phosphor-Imager (Fujifilm, Tokyo, Japan).

S. pombe Mus81-Eme1 and Mus81^DD-Eme1 were purified using an established protocol (Gaskell et al., 2007 (link)). Cleavage reactions, and their analysis by native (10% polyacrylamide) and denaturing (15% polyacrylamide) PAGE, have been described previously (Gaskell et al., 2007 (link); Osman et al., 2003 (link)). The reaction buffer contained 25 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, 100 µg/ml bovine serum albumin, 6% glycerol and 10 mM MgCl₂. Reactions were started by the addition of protein and were incubated at 30°C for 30 min. Reactions were stopped by the addition of one-fifth volume of stop mix (2.5% SDS, 200 mM EDTA and 10 mg/ml proteinase K) and further incubation at 30°C for 15 min to deproteinize the mixture. Reaction products were processed and run on native and denaturing gels as described (Gaskell et al., 2007 (link); Osman et al., 2003 (link)). When mapping cleavage sites on denaturing gels, reaction products were run alongside a Maxam-Gilbert G + A sequencing ladder of the relevant labeled oligonucleotide. A 1.5-base allowance was made to compensate for the nucleoside eliminated in the sequencing reaction. Gels were dried on 3 MM Whatman paper and analyzed with a Fuji FLA3000 PhosphorImager (Fujifilm Corp., Japan).