L arginine hcl

Enzyme activities were measured in supragingival, subgingival and saliva samples (collected in the first recruitment, n = 15 from each group). urease and ADS activities were determined as described by Nascimento et al.^{16 (link)}. In brief, ammonia generated from incubation (37 °C, 120 min) of 125 μl oral samples (suspended plaque and saliva samples) and a 500 μl mixture [50 mM urea (Sigma-Aldrich, St. Louis, MO, USA) or L-arginine-HCL (Sigma-Aldrich), 0.5 mM Tris-maleate buffer (pH = 6.0)] were measured by Nessler’s reagent (Sigma-Aldrich) with ammonium sulfate as a standard. Meanwhile, protein content in each sample was determined by Bradford’s Assay with bovine serum albumin as a standard. urease and ADS activities were expressed as μmol ammonia produced per min and were normalized to mg of protein (μmol/min/mg). LDH activities in plaque and saliva samples were measured by the LDH Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions. One unit of LDH was defined as the amount of the enzyme that catalyzed the conversion of lactate into pyruvate to generate 1.0 μmol of NADH per min. LDH activity was expressed as the amount of enzyme (U) per g of protein (U/g).

The HLA-B*57:01, HLA-B*57:03, HLA-B*58:01 and β₂m genes were sub-cloned into the pET-30 expression vector and were expressed into inclusion bodies separately in Escherichia coli. The HLA complexes were refolded in the presence of the peptides listed in Table 1 and purified as described previously^{63 (link)}. Briefly, 90 mg HLA heavy chain was refolded by rapid dilution in a solution containing 3 M urea (Sigma-Aldrich, USA), 100 mM Tris-HCl, pH 8.0 (Sigma-Aldrich, USA), 400 mM l-arginine-HCl, 5 mM reduced glutathione (Sigma-Aldrich, USA) and 0.5 mM oxidised glutathione (Sigma-Aldrich, USA) in the presence of 30 mg β₂m and 10 mg of the appropriate peptide for 48 h. The refolded HLA-peptide complexes were dialysed into 10 mM Tris, pH 8.0, and purified by size-exclusion chromatography using HiLoad 16/60 Superdex 200 pg (GE Healthcare, USA) columns on an AKTA Purifier (GE Healthcare, USA) FPLC chromatography systems in 10 mM Tris, pH 8.0, and 150 mM NaCl buffer. Final purification was by anion exchange using a HiTrap Q Fast Flow column (GE Healthcare, USA) on the same AKTA system in 10 mM Tris pH 8.0 buffer with a NaCl gradient from 0 to 500 mM over 45 min.

SH-SY5Y cells were purchased from Sigma Aldrich, and were cultured in 100% Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin-streptomycin (pen-strep) at 37 °C and 5% CO₂.
For SILAC labelling of SH-SY5Y cells, the cells were passaged for >14 days in SILAC medium (Thermo-Fisher Scientific) supplemented with 10% (vol/vol) dialyzed foetal calf serum (10,000 MW cut-off, Gibco) and “heavy” amino acids (L-arginine-¹³C₆¹⁵N₄:HCl [50 mg/litre] and L-lysine-¹³C₆¹⁵N₂:2HCl [100 mg/liter]; Cambridge Isotope Laboratories), supplemented with 100 U/ml penicillin-streptomycin, or in the same component SILAC media except with “light” amino acids (L-Arginine:HCl and L-Lysine:HCl, Sigma Aldrich).
To create stably expressing clonal cell lines of CHC22.GFP and CHC17.GFP, the method used was the same as described previously in (Nahorski et al.)⁷ .