Centricon 10

Flag-tagged mouse MEX3C-2 (different from human MEX3C-2 by three amino acid residues) was transiently expressed in HEK293 (ATCC, Manassas, Virginia) by Fugene 6 (Roche)-mediated transfection of pFlag-MEX3C-2 plasmid DNA. Cells from five 15-cm dishes with or without MEX3C-2-Flag expression were used for the protein complex purification. Cells were lysed with 10 ml NP-40 buffer and 0.5 ml anti-Flag agarose beads (Sigma) was added to each pre-cleared lysate for the affinity purification. After washing with 10 ml NP-40 lysis buffer, the protein bound to the beads was eluted with ten 0.5 ml aliquots of 0.1 M glycine HCl (pH 2.5). The presence of MEX3C-2 in the eluent was examined by SDS-PAGE followed by Western blotting analysis with an anti-Flag antibody (Sigma). Flag-tag positive fractions from MEX3C-2 expressed preparation were pooled. Corresponding fractions from control preparations were similarly pooled. The two samples were concentrated to the same volume by spinning the samples in Centricon 10 (Millipore). The proteins in the samples were separated on an 8~16% gradient Ready-gel (Bio-Rad) and stained with Coomassie blue. Bands unique to the MEX3C-2-Flag preparation were recovered from the gel, treated with trypsin, and subjected to LC-MS/MS analysis to identify the identities of the proteins (analyzed by the Biomolecular Resource Facility of Wake Forest University).

Protein A chromatography was carried out as previously described (14 (link), 15 (link)). Four-tenths milliliter aliquots of plasma were adjusted to pH 8.0 by adding 0.8 mL 100 mmol/L Tris (pH 8). After syringe filtration to clarify samples, 1 mL was applied to a 1 mL column of packed protein A beads (Pierce Chemical Co., Rockford, IL, USA) equilibrated in 100 mmol/L Tris, pH 8.0. The column was washed and eluted as previously described (15 (link)). The eluate fractions containing nearly all the recovered protein were pH neutralized and stored at 0–4°C. Inhibitory activity in protein A eluate fractions was unchanged, appearing in the retentate fraction after dialysis (10 mmol/L phosphate, pH 7.4) and ultrafiltration on a 10 kDa cutoff membrane (Centricon-10; Millipore Corp., Bedford, MA, USA). All fractions were sterile filtered (Millipore Corp. Bedford, MA, USA; 0.2 um) before assay for growth-promoting activity.

The synthesis of 2–5A refers to the method reported by Kerr et al.^{21 (link)}. The reaction system of 2–5A (p3(A2′p)n A, n = 1 to ≥3) was as follows: OAS1 buffer (20 mM HEPES (pH 7.6), 20 mM Magnesium acetate, 20 mM KCl, 1 mM EDTA), 20 μg/ml 2′, 5′-OAS1 (Abnova, USA), 1 μg/ml poly (I:C) (InvivoGen, USA), and 10 mM ATP (pH7.4) (CST, USA). The mixture was incubated at 37 °C for 24 h, filtered by Centricon-10 (Millipore, USA), and then centrifuged at 3000 × g for 15 min. The analysis of 2–5A synthesis was carried using a Mono Q HR 5/5 column (GE Healthcare, USA). A 200 μL aliquot was applied to the column equilibrated in Buffer A (20 mM Tris–HCl, pH 7.5) and fractionated in a linear gradient of 0–20% and 20–22.8% of Buffer B (1 M NaCl, 20 mM Tris–HCl pH = 7.5) using 18 column volumes (CV) and 50 CV, respectively. Concentration of the oligoadenylates were determined by application of small aliquots (~20 μl) to the Mono Q HR 16/10 column. For concentration determination, ATP was used as an internal control. 2–5A was diluted by Opti-MEM (Invitrogen, USA) and transfected by using calcium phosphate coprecipitation.

To measure MMP3 activity, conditioned medium from treated cells was concentrated ~20-fold using Centricon-10 spin concentrators (Millipore, Billerica, MA, USA). Samples were quantified by Bradford analysis and equal amounts of protein were mixed with Laemmli sample buffer without reducing agents, incubated for 15 min at 37 °C, and separated on precast gradient SDS-polyacrylamide slab gels containing 1 mg/mL casein (Sigma). Following electrophoresis, the gels were placed in 2.5% Triton X-100 for 30 min, then incubated at 37 °C in 50 mM Tris–HCl, pH 7.4, containing 5 mM CaCl₂ for 18 h. MMP3 bands were visualized by Coomassie blue staining and the level of MMP3 activity was quantified, as previously described^{21 (link)}.