Protease inhibitor cocktail tablet

PMs were prepared from control HEK 293FT cells and various GABA_AR variants were detached using Hanks’ balanced salt solution (Gibco, Waltham, MA, USA) without divalent cations (i.e., trypsin was not used), and the cells were centrifuged at 300× g for 3 min. The HEK 293FT cells or brain (mostly cortex) were homogenized in an ice-cold buffer containing 0.3 M sucrose, 0.5 mM EDTA-Tris, HEPES-Tris, 10 mM (pH 7.3), and protease inhibitor cocktail tablets (A32955, Thermo Fisher Scientific, Waltham, MA, USA), and centrifuged at 10,000× g for 15 min at 4 °C, after which the pellet was discarded. The supernatant was centrifuged for 1 h at 150,000× g and the resulting pellets were resuspended in 20 mM HEPES-Tris pH 7.3. This plasma membrane-enriched preparation was used for further measurements of the enzyme activity. Ethylenediaminetetraacetic acid (60-00-4), 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES), and Tris(hydroxymethylamino-methane (77-86-1) were obtained from Merck (USA).

Protein extraction from B-lymphocytes and monocytes was performed using a T-PER reagent (cat. # 78510, Thermo Fisher Scientific, Waltham, USA) with the addition of protease inhibitor cocktail tablets (cat. # 04693116001, Roche, Basel, Switzerland). Protein concentration was determined using a bicinchoninic acid (BCA) assay (cat. # 23227, Thermo Fisher Scientific, Waltham, MA, USA).

Protein extraction from SHEP and U2OS cells was performed +/- QKI knockdown using a T-PER reagent (cat. # 78510, Thermo Scientific) with addition of protease inhibitor cocktail tablets (cat. # 04693116001, Roche). 100 μL of the reagent was added to the 1 × 10⁶ cells followed by homogenization and sonication (20 sec) for membrane disruption. Cells were centrifuged for 5 min at 10,000 × g to remove cell debris. Protein concentration was determined using a bicinchoninic acid (BCA) assay (cat. # 23227, Thermo Scientific).

VLPs were isolated as described previously [54 (link)]. Briefly, 5 g of leaves were ground in liquid nitrogen and added to 20 ml of 0.1 M sodium phosphate (pH 7.2) supplemented with protease inhibitor cocktail tablets (Thermo Fisher Scientific, A32955). The homogenate was filtered through Miracloth followed by centrifugation to remove plant debris. Supernatant (10 ml) was added to an ultracentrifuge tube (Beckman, 331374) on top of 1 ml of 25% sucrose and 0.5 ml of 70% sucrose in 0.1 M sodium phosphate (pH 7.2). Ultracentrifugation was performed at 40,000 rpm for 2.5 h at 4°C. The 70% sucrose fraction was collected and treated with protein concentrator (Thermo Fisher Scientific, Cat 88513) to exchange the PBS buffer. The solution (5 μl) was applied onto a formvar-coated grid for 10 min and wicked to a thin layer of liquid. Five microliters of 2% glutaraldehyde was applied for 30 s, and liquid was removed with bibulous paper. The sample grid was washed with 5 μl of double distilled water by wicking off the liquid. Five microliters of 2% aqueous uranyl acetate was applied for 20 s and wicked to dryness. Grids were examined and imaged in a Hitachi SU-3500 scanning electron microscope.

All solutions were pre-chilled to 4°C and all steps were carried out on ice to prevent internalisation. Fresh membrane impermeable Sulpho NHS-SS-Biotin (21331, Thermo Fisher Scientific) was dissolved in PBS at a final concentration of 0.2 mg/ml. Neurons were washed twice in PBS before being incubated with biotin for 15 mins at 4°C. The cells were then washed in PBS before being quenched in quenching buffer (50 mM Triz, 100 mM NaCl, pH 7.5) for 10 min at 4°C. The cells were lysed in 2% Triton-X-100 (X100, Sigma) plus protease inhibitor cocktail tablets (A32953, Thermo Fisher Scientific) in PBS and a BCA assay (23225, Thermo Fisher Scientific) carried out to determine protein concentration. Equal protein amounts of lysate were incubated with streptavidin beads (17-5113-01, GE Healthcare) for 1 hr at 4°C before being washed and analysed by western blotting.

Colonic mucosa was homogenized with phosphatase and protease inhibitor cocktail tablet (cat#A32959, Thermo-Scientific, USA) dissolved in RIPA buffer (Cat# 20-188, Millipore, St. Louis, MO, USA) with zirconium beads (2 mm, Cat# 11079124zx, Biospec, Bartlesville, OK, USA) in Precellys 24 tissue homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France). Then, the resultant homogenate was centrifuged at 1000 × g at 4°C. After centrifugation, the supernatant was transferred into a fresh microcentrifuge tube with gentle mixing on the tube rotator overnight at 4°C. The homogenate was centrifuged at 15,000 × g at 4°C for 30 min, the supernatant was diluted (1 : 3) with RIPA buffer, and its protein concentration was estimated using Pierce BCA Protein Assay Kit (Cat# 23225, Thermo-Scientific, USA). Undiluted homogenate was kept in aliquots at -80°C for later use. TNF-α, IL-1β, IL-6, and IL-17 cytokines were determined by ELISA assay in the colonic mucosal homogenates according to the manufacturer's instructions.