ImmunoBlot

Sciatic nerves from all groups in both studies were homogenized in a buffer containing protease inhibitor cocktails (Calbiochem, cat. no. 524624 and 539131) plus 1.0 mmol/L HEPES (Gibco, cat. no. 15630-080), 5.0 mmol/L benzamidine, 2.0 mmol/L 2-mercaptoethanol (Gibco, cat. no. 21985), 3.0 mmol/L EDTA (Omni pur, cat. no. 4005), 0.5 mmol/L magnesium sulfate, 0.05% sodium azide; final pH 8.8 as previously described (7). Samples were pre-cleared by centrifugation at 5,000 × g for 5 min at room temperature. Supernatants were retained as whole lysate and stored at −80°F until use.

After determination of protein content by bicinchoninic acid assay (Thermo Fisher Scientific, cat. no. 23225), lysates were loaded (20 μg total protein/lane) on 4–12% Bis-Tris gels (Invitrogen, cat. no. WG1402BX10) and electrophoresed in 5% HEPES running buffer and transferred onto PVDF membrane with iBlot transfer stacks (Invitrogen, cat. no. IB24001) using NuPage transfer buffer (ThermoFisher Scientific, cat. no NP0006). The membranes were blocked in 5% BSA in phosphate-buffered saline-tween 20 (PBST) for 1 h. Membranes were incubated overnight at 4°C with primary antibodies (TFAM, ThermoFisher Scientific, cat# PA5-23776; Total OxPhos Complex Kit, ThermoFisher Scientific, cat# 458099; DNM1L Santa Cruz Biotechnology; sc-32989). Following visualization, blots were stripped and probed with a mouse monoclonal antibody against β-actin (ACTB; Sigma Aldrich, cat. no. A5441) in blocking buffer as a loading control. All blots were washed in PBST then incubated with species-specific IgG conjugated to HRP (American Qualex, cat. no. A102P5) diluted 1:5,000 in PBST and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, cat. no. 34096). Images of protein bands were analyzed by semi-quantitative analysis using the VersaDoc gel imaging system and Quantity One software (Bio-Rad). The densitometry of TFAM, OXPhos, and DNM1L bands were normalized to densitometry of ACTB.