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).
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).