Biomarkers levels were measured in duplicate using a commercial kit according to manufacturer's instructions: IL-6, IL-1-beta, and IL-10 (Siemens Medical Solutions Diagnostics, Caernarfon, UK) in an Immulite analyzer, and CRP (Roche Diagnostic, Basilea, Switzerland) in a Hitachi 917 analyzer.
After 24 h the cytokines in the supernatant were analyzed with a cytometric bead array for human Th1/Th2 cytokines (BD Biosciences, San Diego, CA) according to the manufacturer's instructions (detecting IL-2, IL-4, IL-5, IL-10, TNFα and IFNγ).
Kv12.2 channel membrane expression was assessed by membrane surface biotinylation assay utilizing a 'Cell surface protein isolation kit' (Pierce, IL), according to instructions from the manufacturer.
NF-κB, or p53 EMSA was performed according to The Thermo Scientific LightShift Chemiluminescent EMSA Kit (Rockford, IL) following the instructions of the manufacturer.
Cytokine levels were analyzed via ELISA according to the manufacturer's instructions (IL-5, IFN-γ (BD Pharmingen) and IL-13 (R&D Systems Minneapolis, MN)).
Protein levels of IL-10, IL-6, TNF-alpha, IFN, and BDNF were assessed by sandwich ELISA, using a commercial kit according to the manufacturer's instructions (Cedarlane Burlington, Canadaa): IL-10 #CL76130, IL-6 #CL76126K, TNF-alpha #CL76145K, IFN #CL76120K; Millipore (Billerica, MA, USA): BDNF #CYT306).
Concentrations of IL-1β, IL-6, IL-8, and TNFα in cell culture supernatants were measured by ELISA according to the manufacturer's instructions (Bovine IL-1β Screening Set ESS0027 and Bovine IL-6 Screening Set ESS0029 [Thermo Scientific] and Bovine TNFα DuoSet DY2279 and Human CXCL8/IL-8 DY208t DY208 [R&D Systems Europe Ltd]).
Cytosolic and nuclear protein fractions were isolated through NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL) following manufacturer's instructions and HDAC activity assays (Cayman Chemical, Ann Arbor, MI) were performed as per manufacturer's instructions.
Total IgG was purified from 0.5 ml of serum samples from eleven randomly selected NC patients using Immobilized Protein G Plus (Pierce, Rockford, IL) according to manufacturer instructions.
The bound fraction was eluted as described above, and used for Fab fragments production with a commercial kit (ImmunoPure Fab Preparation Kit - Pierce, Rockford, IL), following manufacturer's instructions.
