Ndufa9

Mitochondria were isolated from B cells using Mitochondria isolation kit (Miltenyi Biotec)
following manufacturer's instructions with some modifications. Briefly, frozen B cells
(2–10 × 10⁷ cells) were directly resuspended in pre-cooled phosphate-buffered
saline (1 ml per 10⁸ total cells) supplemented with ethylenediamine tetraacetatic
acid (EDTA; 2 mM), anti-protease and –phosphatase inhibitors and benzonase
(50 U), and incubated for 20 min at 4 °C. Cell homogenization was performed
with a 26-G needle stepwise using 5–10 stokes. Lysates were diluted to 1 × separation
buffer (Miltenyi Biotec) and proceeded to magnetic labeling by incubation with anti-Tom22 magnetic
beads for 1 h at 4 °C on a wheel. The labeled cell lysate was loaded on a MACS
column placed in a magnetic field and let run through. Lysates were re-loaded three times. Column
was then intensively washed out and the magnetically labeled mitochondria were then flushed out.
Pre- and post-mitochondria-purified fractions were separated by SDS-PAGE and the presence of
cytoplasmic β-actin protein, Mt hsp60 or NDUFA9 proteins, and nuclear KDM1a were
evaluated by western blot analysis using monoclonal anti-β-actin (AC-15,
Sigma-Aldrich), -hsp60 (Biosciences Inc., Allentown, PA, USA), -NDUFA9 (Abcam, Cambridge, UK) and
KDM1a (Cell Signaling Technology).

Mitochondrial primary antibodies: TOMM20 (Santa Cruz), TIMM9 (Abcam), NDUFA9 (Abcam), COX4i1 (Abcam), ATP5A (Abcam), β-ACTIN (Sigma-Aldrich). Horseradish peroxidase (HRP)-linked secondary antibodies were used (Sigma-Aldrich). LC3 primary antibodies: Rabbit anti-LC3 (Novus Biologicals; NB100–2220) used at 1:1000 dilution; mouse anti-actin (Sigma A5316) used at 1:500 dilution. Secondary antibodies: Polyclonal goat anti-rabbit immunoglobulins/HRP (Dako P0448) used at 1:5000; polyclonal goat anti-mouse immunoglobulins/HRP (P0447) used at 1:5000. Beta- and Gamma-synuclein primary antibodies: mAb α/β-Synuclein (Syn205, Cell Signaling; 1:1000) or pAb γ1-Synuclein (1:1000). γ1-Synuclein polyclonal antibody was raised to the peptide DFSHGGMEGGEGGEGY by immunization of rabbits (New England Peptide) and affinity purified as previously described (54 (link)). IRDye-800 and IRDye-680 (LI-COR, Lincoln, NE) conjugated secondary antibodies (1:10 000) enabled the blot to be imaged using an Odyssey Infrared Imager (catalog no. 9120; LI-COR) with a wide linear range.

T cells were lysed in Ripa buffer containing a protease inhibitor and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA). Cell lysates were centrifuged at 4 °C at 12,000× g for 10 min, separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% BSA in PBS containing 0.05% Tween-20 and incubated with various antibodies recognizing pAMPKα1 (T₁₇₂), pAMPKα1 (S₄₈₅), pS6 (S_235/236), peIF4E (S₂₀₉), pULK1 (S₅₅₅), ATG7, AQP9, PGC1α, TFAM, OPA1, pDRP1 (S₆₁₆), ID2, ID3, cMyb, cMyc, Blimp1, T-bet, ZEB2, FOXO1, TCF1, TRAF6, pTSC2 (S₁₃₈₇), Eomes, HIF-1α and β-actin (Cell Signaling Technology) and the Complex I subunit NDUFA9 (Abcam, Cambridge, MA, USA). The membranes were imaged using a BioRad Chemidoc MP (Bio-Rad, Hercules, CA, USA) after a second incubation step with a horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Cell Signaling) secondary antibody [18 (link)].

Reagents: 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO) (Cayman Chemical) and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Sigma-Aldrich), cycloheximide (Sigma-Aldrich), oligomycin (Sigma-Aldrich), MKT-077 (Sigma-Aldrich). Antibodies for Western blot analysis: LONP1 (Proteintech, 1:3000), OXA1L (Proteintech 1:10000), mtHSP70 (Proteintech, 1:10000), GRPEL1 (Proteintech, 1:5000), TIM44 (Proteintech, 1:5000), CLPX (Proteintech, 1:3000), VDAC1 (Abcam, 1:3000), NDUFA9 (Abcam, 1:3000), DNAJA3 (Santa Cruz BioTech, 1:1000), HSP60 (Santa Cruz BioTech, 1:3000), TOM20 (Santa Cruz BioTech, 1:3000), anti-FLAG M2 (Sigma-Aldrich, 1:10000), anti-HA.11 (Covance, 1:5000).