Anti flag m2 affinity agarose

For CIC immunoprecipitation, myc-CIC-S or rabbit/mouse IgG (control) antibody-bound beads were prepared by incubating anti-CIC antibody, anti-myc antibody, normal rabbit-IgG, or mouse-IgG with protein G agarose beads (Roche) in IP buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA with 1X complete protease inhibitor from Roche) at 4°C overnight. Cell lysates were first pre-cleared with Sepharose 4B beads (Sigma) at 4°C for 1 hour and the lysates were immunoprecipitated with the prepared protein-G beads at 4ºC for 4 hours. The captured proteins and protein complexes were released by boiling the beads in 2X LDS Sample Buffer (Invitrogen) at 95^oC for 5 minutes or 2X SDS sample buffer (117mM Tris-Cl pH6.8, 4% SDS, 8% glycerol, 0.01% bromophenol blue, 200mM DTT) at 98^oC for 10 minutes (see supplementary for details). For immunoprecipitations using FLAG epitopes, complexes in the pre-cleared lysate were captured by anti-FLAG M2 Affinity agarose (Sigma) for 2 hours at 4°C. Captured proteins and complexes were eluted by two rounds of FLAG-peptide competition (400ug/mL FLAG peptide, 50mM ammonium bicarbonate) for 30 minutes at 4°C.

All FRET peptides reported in this study were custom-synthesised by Bachem (Switzerland). Aliphatic index and Grand average of hydropathicity (GRAVY) were computed using ProtParam (https://web.expasy.org/protparam/). Recombinant human pro-MMP-2 and ADAM-17 were purchased from Calbiochem (Merck-Millipore). Pro-MMP-12 was purchased from R&D Systems (Cat. n.: 917-MP). Expression and purification of recombinant human ADAMTS-1, −4, −5 and −7 have been reported before^8–10 (link). ADAMTS-7 used in this study was the recombinant construct ADAMTS7-T8, which lacks the C-terminal PLAC domain and is described in Colige et al⁸ (link) and de Groot et al¹⁰ (link). ADAMTS-8 was transiently expressed in HEK293T and purified with anti-FLAG M2 affinity agarose (Sigma Cat. n: A2220) exactly like ADAMTS-1, ADAMTS − 4 and ADAMTS − 5. ADAMTS-1, ADAMTS − 4, ADAMTS − 5 and ADAMTS − 8 constructs had a C-terminal FLAG-tag (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Enzyme concentrations were determined by active site titration⁹ (link)^,11 (link). The mammalian expression vector for LTBP4S-A, containing an N-terminal FLAG tag was a generous gift of Tomoyuki Nakamura (Kansai Medical University, Osaka, Japan) and details of its generation have been described previously¹² (link). Recombinant human TIMP-4 was purchased from R&D (Cat. n: 974-TSF).

Various combinations of expression vectors were co-agroinfiltrated into N. benthamiana leaves and samples were harvested at 2.5 days post agroinfiltration and ground in a cooled mortar in GEN buffer (10% [v/v] glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.5% [v/v] Triton X-100 and protease inhibitor cocktail). The supernatants were incubated with anti-Flag M2 affinity agarose (Sigma-Aldrich) in Bio-spin chromatography columns (Bio-rad) for 2 h at 4°C on a rotator, followed by washing with the washing buffer (10% [v/v] glycerol, 25 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, 1mM DTT and 0.1% [v/v] Triton X-100). Elution of purified proteins was as described above [137 (link)].

Cellular extracts were pre-cleared using Protein A agarose beads (Sigma-Aldrich) for 1h at 4°. Afterward, 200 µl of extract were incubated with 20 µl anti-FLAG M2 affinity agarose (Sigma-Aldrich) for 2h at 4° with gentle mixing. This was followed by washing the bead material three times with 300 fresh B70+Inhibitors. RNA was isolated from the matrix material as well as the input as described below.