Halt protease inhibitor mixture

Western blotting was conducted as described previously (26 (link)). Briefly, the cells were lysed at the indicated times with mammalian protein extraction reagent (Thermo Fisher Scientific) that included protease inhibitor mixture (halt protease inhibitor mixture, Thermo Fisher Scientific). Lysates were centrifuged at 15,000 r.p.m. for 10 min (4 °C). Cell protein lysates (∼100 μg) were dissolved in loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.01% bromophenol blue, 100 mM DTT) and separated by SDS-polyacrylamide gel electrophoresis. Antibodies used were SLC9A9 Polyclonal Antibody (PA5-42524, Thermo Fisher Scientific) at 1:50 dilution and monoclonal α-tubulin antibody (T 9026, Sigma) at 1:500 dilution.

The different stable cancer cell lines (1 × 10⁶ cells) suspended in 500 μl of serum-free DMEM supplemented with insulin-transferrin-selenium (Life Technologies) were injected subcutaneously into the backs of 8-week-old female athymic mice (Janvier). Tumor dimensions were measured at least twice a week using calipers and the tumor volume was determined by using the formula: (4π/3) × L/2 × W/2 × H/2, (L, length; W, width; and H, height). When the tumor volume reached 1000 mm³, mice were euthanized and the tumors were excised. For protein analysis, tumors were lysed directly after harvesting. Tumors were incubated in cell extraction buffer (FNN0011, Thermo Scientific) supplemented with Halt protease inhibitor mixture (78429, Thermo Scientific) and lysed using a Precellys homogenizer. Animal housing was done in compliance with the European Union directive 2010/63/EU. Briefly, each cage contained five mice with an enriched environment, food and water were given ad libitum, and the litter was changed on a weekly basis. Animal care met the European Union directive 2010/63/EU ethical criteria. The animal experimentation protocol was approved by the local animal care committee. (Veterinary service and direction of sanitary and social action of Monaco Dr. H. Raps.)

DNA encoding GFP binding protein (GBP)52 (link) (synthesized by Genscript) was inserted into pET24B vector. Recombinant His6x-tagged GBP was purified on Talon resin (Clontech), and then conjugated to high density glyoxal agarose beads (Agarose Bead Technologies). GBP beads were used for IP similar to agarose protein G beads. Cells were collected by trypsinization (HeLa) or by gentle resuspension (HEK293T), and lysed in 50 mM Tris, pH 7.4, 150 mM NaCl; 1% Triton X-100 supplemented with Halt protease inhibitor mixture (Thermo Scientific) and 1 mM PMSF for 1 hour on ice.
Cell lysate was spun at 20,000 × g and supernatant was added to 20–25 μL of 50% GBP-bead suspension. Bead-lysate mix was incubated for 2 h at room temperature while rotating. Unbound material was removed and the beads were washed three times in 0.05% Triton X-100 in PBS and eluted in 2X sample buffer to a total volume of 50 μL.

For the cytokine analysis, a portion of each tumor was lysed and homogenized with radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher) containing Halt protease inhibitor mixture (Thermo Fisher) in a Fast Prep tissue homogenizer (MPBio). After lysis and homogenization, protein concentration was determined by using a bicinchoninic acid assay kit (Thermo Fisher). Each lysate (1 μg/well) was analyzed for murine MCP-1, MIP-1α, and MIP-1β by MSD multiplex assay.