Tpck treated bovine trypsin

Groups of six midguts were homogenized in assay buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM CaCl₂, pH7.6), and homogenates were centrifuged at 10,000 g (4°C) for 10 min. The supernatants were collected and stored at −80°C for activity assays. MMP activity of midgut samples was measured using the generic MMP assay kit (Anaspec, Fremont, CA, USA). Each sample/treatment was analyzed as three independent biological replicates.
Recombinant AeLT and AeSP1 were activated with trypsin according to the method of Tsu and Craik [9 (link)]. The activation reaction contained 5 μl of recombinant protein (20 ng), 2.5 μl of 1 mM TPCK-treated bovine trypsin (Sigma) and 2.5 μl of 4x assay buffer. Following activation at RT for 15 min, FS-6 substrate (Millipore) was added to a final concentration of 10 μM in reaction buffer and fluorescence intensity was measured at Ex = 328 nm and Em = 393 nm every 20 min (over 4 h) with a Perkin-Elmer LS50B spectrometer. For rAeTIMP inhibition assay, 20 ng of the active recombinant AeLT and AeSP1 were preincubated with 20 ng of rAeTIMP at RT for 2 h before adding the fluorescent substrate.

Cross-linked samples in 10 mM sodium phosphate buffer pH 8.0 for the GADD45β/MKK7_KD complex or in 50 mM ammonium bicarbonate buffer pH 8.0 for the DTP3/MKK7_KD complex were reduced at 55 °C for 1 h by adding dithiothreitol (DTT) up to 10 mM final concentration. Following carbamidomethylation with Iodoacetamide (IAM, 7.5 mM final concentration) at room temperature in the dark for 15 min, enzymatic hydrolyses were performed with TPCK-treated bovine trypsin (Sigma Aldrich) at 37 °C for 24 h with an enzyme-to-substrate ratio of 1:50 (w/w). After digestions, samples were centrifuged at 10.000 ×g for 15 min and supernatants were diluted in H₂O/Formic acid 0.1% at a final concentration of 500 fmol/μL for MS analysis.