*2.1. Patients*

Fifteen patients with MS according to McDonald criteria [27] and fifteen healthy volunteers control subjects (HC) were enrolled into the study. The patients and HC subjects were selected by the Centre of Multiple Sclerosis at Department of Basic Medical Sciences Neurosciences and Sense Organs, University of Bari. All patients had to be without any immunomodulatory treatment at least 6 months prior to study entry. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Ethics Committee of Azienda Policlinico di Bari (Project identification code 5275). Table 1 reports demographic and clinical characteristics of MS patients and healthy subjects. For all data, no significant di fference was observed between males and females as well as between RR and SP forms of MS.


**Table 1.** Demographic and clinical characteristics of patients with multiple sclerosis (MS) and healthy control subjects (HC) enrolled into the study. (SP: secondary progressive, RR: relapsing-remitting, EDSS: Expanded Disability Status Scale, SEM: standard error of mean.

### *2.2. Sample Preparation*

Peripheral blood mononuclear cells (PBMCs) were isolated from K3-EDTA blood by centrifugation on a Ficoll-Hypaque density gradient (density: 1.077 g/mL; Amersham Pharmacia Biotech, Buckinghamshire, UK), washed twice and resuspended in PBS. Total protein concentration was determined by Bio Rad protein assay.

### *2.3. Electrophoretic Procedures and Western Blotting*

Protein of PBMCs were resuspended in RIPA lysis buffer, separated by 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred into a nitrocellulose membrane. The membrane was blocked with 5% fatty acid free dry milk in 500 mM NaCl, 0.05% Tween 20, 20 mM Tris, pH 7.4 (TTBS) for 3 h at 4 ◦C and probed over night with antibodies against OPA1 (whole molecule of OPA1 protein was used as the immunogen) (Thermo scientific, Pierce Antibodies, Lausanne, Switzerland), PHB2 (Invitrogen, Paisley, UK), OMA1, SIRT3 (Cell Signalling, Danvers, MA, USA) and β-actin (Sigma-Aldrich, St. Louis, MO, USA). After being washed in TTBS, the membranes were incubated for 60 min with anti-rabbit or anti-mouse IgG peroxidase-conjugated. Immunodetection was then performed, after further TTBS washes, with the enhanced chemiluminescence (ECL) (Euroclone, Paignton, UK). Densitometric analysis, expressed as arbitrary densitometric units (ADU), electrophoretic profile and relative front (Rf) determinations were performed by Image Lab Touch 2.4 software (BioRad, Milan, Italy). Rf indicates the relative movement of the band from the top.
