Anti opa1

PBMCs were plated in a 96-well flat-bottom plate and cultured in RPMI-1640 supplemented with 10% FBS at a density of 2 × 10⁶ cells per well. Cells were stimulated with 96-well plates coated with anti-CD3 (Clone: OKT3; 2 µg/mL, Biolegend, San Diego, CA, USA) or PBS control and measured at 4 h and 24 h timepoints at 37 °C/5% CO₂. After stimulation, cells were stained with cell surface markers anti-CD3, -CD4, -CD27, -CD28, -CD45RO and -CD45RA, permeabilized and then stained with anti-GLUT1, anti-OPA1, anti-MFN2 and anti-DRP1 (Abcam, Cambridge, UK). Cell viability was assessed using Live/Dead Aqua (ThermoFisher, Waltham, MA, USA). Cells were analysed using the BD LSRFortessa^TM X-20 cell analyser (BD Biosciences, Franklin Lakes, NJ, USA). Single-stained BD CompBeads were used to define compensation parameters. T-cell subsets were identified using the gating strategy observed in Figure 1.

Western blotting was carried out as described before.^{22 (link)} Total protein was extracted by RIPA buffer containing protease cocktail inhibitor. All protein samples were quantified. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% skim milk in TBS-T (BD Pharmingen), PVDF membranes were incubated with the primary antibody overnight at 4 °C and then with the secondary antibody for 1 h at room temperature. The following antibodies were used: anti-MFN1 (1:1000, Abcam), anti-MFN2 (1:1000, Abcam), anti-Opa1 (1:1000, Abcam), anti-DNM1L (1:1000, Abcam), anti-MFF (1:1000, Abcam), anti-TOM20 (1:1000, Abcam), anti-E-cadherin (1:1000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Snail (1:1000, Abcam), anti-Twist (1:1000, Abclonal), anti-Zeb1 (1:1000, Abclonal), anti-Slug (1:1000, Abclonal) anti-Vimentin (1:1000, Abcam) and anti-β-actin (1:1000, Abcam). Protein bands were detected by using image acquisition using ImageQuant™ LAS 4000 (GE Healthcare Life Sciences).

Western blotting was used to assess the protein expression and phosphorylation as described previously [28 (link)]. The immunoblots were incubated with primary antibodies against anti-PI3K, anti-Akt, anti-p-Akt (Ser473), anti-Bcl2, anti-Bax, anti-Drp1 (Cell Signaling Technology), anti-Mfn1, anti-Mfn2, anti-Opa1 (Abcam), or anti-Fis1 (Genetex) overnight at 4 ? followed by incubation with the corresponding secondary antibodies at room temperature for 1-2 h. The raw bands were detected and quantified with the Bio-Rad imaging system (Bio-Rad, CA, USA).

BMDM were infected (10⁶ cells/time post-infection; 1 PFU/cell) with VACV and collected at the indicated times post-infection. Cell extracts were obtained using lysis buffer (50 mM Tris-HCl, 0.5 M NaCl, 10% NP-40, 1% SDS) and protein extraction was performed for 5 min on ice. Protein lysates (100 μg) were fractionated by 12% or 7.5% SDS-PAGE, transferred to nitrocellulose membranes, and incubated with the following primary antibodies: anti-ISG15, anti-Arg-1 and anti-iNOS (Santa Cruz Biotechnology), anti-actin and anti-tubulin (Cell Signaling Technology), anti-ATG3, anti-ATG5, anti-ATG7, anti-LC3B and anti-SDHA (Novus Biologicals), anti-OPA-1, anti-CORE2 and anti-NDUFA9 (Abcam). Secondary antibodies were goat peroxidase conjugates (Santa Cruz Biotechnology) and mouse and rabbit peroxidase conjugates (Sigma). Protein expression was detected using enhanced chemiluminescence (ECL) reagents (Amersham). For the quantification Image J software was used and density of the specific band related to actin was represented.

Crude homogenates suitable for immunoblot analysis were obtained by homogenizing one brain hemisphere in 15 mM KCl + 1 mM KH₂PO₄, pH 7.4, at 24,000 rpm/min for 90 sec in the cold, followed by centrifugation at 18,690 × g for 15 min at 4 °C. Proteins from homogenates stored at −80 °C (10 μg) were separated by 8% SDS–PAGE and transferred onto a nitrocellulose membrane (Bio-Rad, Hertfordshire, UK).
The milk-blocked membrane was then incubated overnight at 4 °C with the anti-CS (1:2000), or anti-OPA1 (1:2000), or anti-OMA1 (1:1000), or anti-DRP1 (1:1000) antibodies (Abcam, UK), subjected to a horseradish-peroxidase–conjugated anti-rabbit IgG (1:5000; Roche Diagnostics, Mannheim, Germany), and revealed with an enhanced chemiluminescence (ECL) Western blot analysis kit (Applied Biosystems) or by the alkaline phosphatase technique. ECL films were scanned densitometrically, and the optical density of bands was quantified using Image J version 1.38 software. Values obtained from the immunoblot quantification of CS were either used to obtain a measure of mitochondrial mass^{37 (link), 38} or to calculate the semi-quantitative measure of the different proteins relative to CS.

Antibodies (anti-phospho-mTOR, mTOR, phospho-AKT, AKT, phospho-AMPK, AMPK) were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-OPA1, Protein Phosphatase Inhibitor Cocktail IV, and Protein Protease Inhibitor Cocktail (EDTA-free) were purchased from Abcam (Cambridge, MA, USA). T-PER™ Tissue Protein Extraction Reagent, TRIzol™ Reagent, and M-MLV reverse transcriptase were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-GAPDH antibody was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Bradford's solution and PVDF membranes were from Bio-Rad Laboratories (Hercules, CA, USA). Secondary antibodies were obtained from Calbiochem (Burlington, ON, Canada). Organic and inorganic compounds, acids, and solvents were acquired from Merck (Darmstadt, Germany). Westar Supernova substrate was obtained from Cyanagen (Bologna, Italy). SensiFAST SYBR Hi-ROX was purchased from Bioline Meridian Biosciences (London, UK).