Ab16956

RNA was extracted from osteoclasts using peqGOLD TriFast (PeqLab, Erlangen, Germany) at the indicated time points, reverse transcribed into cDNA and analyzed with SYBR Green-based quantitative Real-Time PCR in triplicates. mRNA expression level was normalized to GAPDH, β-actin or RNA polymerase II Polr2a. GAPDH is commonly used as a reference gene in osteoclast studies and indeed was stable in our experiments. To confirm that it does not affect the results when analyzing genes involved in energy metabolism we also used the mentioned above genes.
Antibodies to the following proteins were used for western blotting: NFATc1 (sc-7294, Santa Cruz), DC-STAMP (MABF39, Millipore), Sirt1 (07–131, Millipore), Sirt3 (#5490, Cell signaling), acetylated Sod2 (acetyl-superoxide dismutase K68, ab137037, Abcam), Sod2 (ab16956, Abcam), phosphorylated AMPKα (AMP-activated protein kinase, #2535, Cell Signaling), AMPKα (#2793, Cell Signaling), phosphorylated ACC (Acetyl CoA Carboxylase, #3661, Cell Signaling), ACC (#3662, Cell Signaling), acetylated p65 (Acetyl-NF-κB p65(Lys310) #3045, Cell signaling), p65 (#3987, Cell signaling), IκBα (#4814, Cell signaling), HSP90 (610419, BD Transduction laboratories) and GAPDH (ab8245, Abcam, UK) were used as reference proteins.

Immunoblot analysis was performed as we described previously30 (link). Rabbit polyclonal anti-ANT-1 antibody and rabbit monoclonal anti-β-actin antibody were from Sigma-Aldrich (St Louis, MO, USA, SAB2105530, 1:1,000) and Cell Signaling (Danvers, MA, USA, 4970, 1:5,000), respectively. Rabbit polyclonal anti-ANT2 antibody was generated by immunization with KLH-conjugated N-terminus ANT2 peptide (NH-TDAAVSFAKDFLAG-COOH) at Lampire Biological Laboratories (Ottsville, PA, USA) and purified with protein G (GE Healthcare, Buckinghamshire, UK) and antigen-conjugated columns. The ANT2 antibody was diluted to 1:200 as being used. COX IV (4850, 1:1,000) and VDAC (4866, 1:1,000) antibodies were from Cell Signaling (Danvers, MA, USA). Tom20 antibody was from Santa Cruz Biotechnology (sc-11415, 1:500), Catalase and SOD2 antibodies were from Sigma (St. Louis, MO, USA, C0979, 1:1,000) and Abcam (Cambridge, UK, ab16956, 1:1,000), respectively. Uncropped scans of the critical immunoblots are shown in Supplementary Fig. 6.

The slides were dewaxed, rehydrated, and rinsed twice in phosphate-buffered saline. Antigen retrieval with HistoReveal (Abcam, Cambridge, UK) was performed as a pretreatment. Slides were incubated with 3% hydrogen peroxide for 10 min and then with Protein Block (Abcam, Cambridge, UK) for 30 min. Sections were incubated with anti-NOS-3 mouse monoclonal antibodies (sc-376751, 1:50, Santa Cruz, Dallas, TX, USA), anti-MnSOD mouse monoclonal antibodies (ab16956, 1:300, Abcam, Cambridge, UK), and anti-MMP-9 mouse antibodies (DuoSet Human MMP-9/TIMP-1 Complex, DY1449, R&D Systems, Minneapolis, MN, USA) at 4 °C for 18 h. The color reaction was performed using biotinylated goat anti-polyvalent, streptavidin peroxidase, and DAB substrate (ab64264, Abcam, Cambridge, UK). Nucleic and other basophilic proteins were counterstained with hematoxylin (ab220365, Abcam, Cambridge, UK). The negative control consisted of samples incubated without the primary antibody. Slides were dehydrated, closed with a glass coverslip using DPX (Avantor Performance Materials Poland S.A, Gliwice, Poland), and examined using an Olympus BX51 microscope.

Proteins were separated by SDS‐PAGE, transferred to a nitrocellulose membrane and blocked in BSA. Primary antibodies were diluted as follows: NFE2L2 (H300) 1:2500 (sc‐13032; Santa Cruz Biotechnology, Inc., Dallas, TX), PGAM5 1:3000, CS 1:5000 and SOD2 1:10000 (ab126534, ab16956, ab96600; Abcam plc, Cambridge, UK). IR dye‐coupled secondary antibodies were detected in a fluorescence scanner (Odyssey Scanner; LI‐COR Biosciences, Lincoln, NE). Quantitative analysis was performed using ImageJ software (Schneider et al., 2012).

Proteins were isolated from microdissected formalin‐fixed, paraffin‐embedded (FFPE) cerebellum sections using the Qproteome FFPE Tissue kit (Qiagen, Valencia, CA) according to the manufacturers' instructions. Subsequent Western blotting of protein lysates and densitometric analysis were carried out as previously described.29 Primary antibodies used were anti‐β actin (ab8227, 1:5,000, Abcam), anti‐catalase (C0979, 1:2,000, Sigma‐Aldrich), anti‐human frataxin (ab110328, 1:2,000, Abcam), anti‐frataxin (ab113691, 1:2,000, Abcam), anti–glutathione peroxidase‐1 (ab22604, 1:1,000, Abcam), anti‐NeuN (ab177487, 1:5,000, Abcam), anti–nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2; sc‐722, 1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti‐peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1alpha) (sc‐13067, 1:3,000, Santa Cruz Biotechnology), anti‐SOD1 (ab16831, 1:5,000, Abcam), and anti‐SOD2 (ab16956, 1:5,000, Abcam). (Of note, increases in total frataxin are likely to be underestimated when compared to human frataxin due to differences in antibody (ab113691, Abcam) species reactivity; the human protein reactivity of the antibody is approximately 50% of mouse protein reactivity at the same concentration. Commercial mouse‐specific frataxin antibodies are not available.)