Anti sod2 mnsod

The liver was lysed in lysis buffer (1 mM EDTA, 150 mM NaCl, 1 mM FH₄N, 10 mM NAM, 0.5 mM DTT, 0.01 mM Trichostatin A) in the presence of protease inhibitors using a tissue lyser. The homogenates were centrifuged. Lysates (20 µg proteins) were loaded on a 10% polyacrylamide gel, and the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Detection was performed with primary antibodies (see below), following incubation with the appropriate secondary antibodies and ECL detection (GE Healthcare, Milan, Italy). Band intensity was quantified with the ChemiDoc imaging system (Bio-Rad, Milan, Italy).
The following primary antibodies were used: anti-CD38 (kindly provided by Prof. Fabio Malavasi, University of Torino, Turin, Italy); anti-vinculin (Cell Signaling Technology, #4650 Danvers, MA, USA); anti-SOD2/MnSOD (acetyl K68) antibody (Abcam, ab137037, Boston, MA, USA); anti-SOD2/MnSOD (Abcam, ab13533); anti-GAPDH (Merck Millipore, G8795, Milano, Italy).

The following flow antibodies were purchase from Biolegend: Antimouse CD45-Brilliant Violet 421; Antimouse Ly-6G-Alexa Fluor 488; Antimouse Ly-6G-Alexa Fluor 647; Antimouse CD31-Alexa Fluor 647; Antimouse CD31-PE/Cyanine7; Antimouse CD45 FITC; Anti-mouse/human CD11b-Brilliant Violet 421; Antimouse CD184 (CXCR4)-PerCP/Cyanine5.5; Antimouse CD182 (CXCR2)-APC/Cyanine7.
The following flow antibodies were purchase from Invitrogen: Anti-CD144 (VE-cadherin)-eFluor 660; Anti-CD54 (ICAM-1)-PE; Anti-CD11b-APC; Antimouse F4/80-PE.
The following flow antibodies were purchase from BD Biosciences: Antimouse Ly6G-PE; Anti-Mouse CD16/CD32. Anti-Fibrin (Cat#MABS2155) was purchased from Sigma-Aldrich; Anti-SOD2/MnSOD (Cat#ab68155) and Anti-CD15 (Cat#ab135377) were was purchased from Abcam; Antirabbit β-Actin-HRP (Cat#AF5006) was purchased from Beyotime; Antirabbit IgG-HRP (Cat#7074 S), anti-Ly6G (Cat#87048 S) and anti-COX IV (Cat#4844 S) were purchased from Cell Signaling Technology; Anti-TOMM22 (Cat#66562-1-Ig) was purchased from Proteintech; InVivoMAb antimouse Ly6G (Cat#BE0075-1) and InVivoMAb rat IgG2a isotype control (Cat#BE0089) were purchase from Bio X Cell; Donkey antirabbit IgG (H + L)-Cy3 (Cat#711-165-152) and donkey anti-mouse IgG (H + L)-Cy3 (Cat#715-165-150) were purchased from Jackson ImmunoResearch.

Samples of rat liver were homogenized as previously described [38 (link)]. Liver lysates containing 30 µg protein were loaded in each lane and were electrophoresed on SDS-PAGE gels and transferred to nitrocellulose membrane. Membranes were blocked and after probed with the following antibodies: anti-PGC1α (Millipore, Burlington, MA, USA), anti-NRF1 (Abcam, Cambridge, UK), anti-TFAM (Abcam), anti-TOM20 (Cell signaling), anti-GPX4 (Abcam), anti-PRDX3 (Abcam), anti-SOD2/MnSOD (Abcam), anti-CAT (Abcam), anti-DRP1 (Abcam), anti-OPA1 (Abcam), anti-MNF2 (Abcam), anti-PAMPK Thr¹⁷² (Cell signaling, Danvers, MA, USA), anti-AMPK (Cell signaling), anti-PULK Ser⁵⁵⁵ (Cell signaling), anti-ULK1 (Cell signaling), anti-AMBRA1 (Cell signaling), anti-PINK1 (Cell signaling ) anti-PARKIN (Cell signaling), anti-LC3B (Novus biologicals), anti-POLγ (Novus biologicals, Bio-Techne SRL, Milano, Italy), anti-Total OXPHOS complexes cocktail (Abcam), and anti-βACTIN (Sigma Aldrich, St. Louis, MO, USA). As secondary antibodies, peroxidase anti-rabbit IgG (Vector Laboratories Burlingame, CA, USA) and peroxidase anti-mouse IgG (Vector Laboratories) were used. Horseradish peroxidase-conjugated secondary antibodies were detected with enhanced chemiluminescence.