Supplementary materials and methods
Drp1 silencing–LipofectamineTM RNAiMax (Invitrogen, Karlsruhe, Germany) withone
of
two
different
siRNA
AAGCAGAAGAATGGGGTAAAT-3`
single
and
sequences
of
Drp1
(5`-
5`-CAATCCGTGATGAGTATGCT-3`;
Dharmacon, Thermo Scientific, Bonn, Germany) or nonfunctional mutant RNA (5`AAG AGA AAA AGC GAA GAG CCA-3`; Dharmacon, Thermo Scientific, Bonn,
Germany) were dissolved in Optimem I (Invitrogen, Karlsruhe, Germany) according
to the manufacture`s protocol of reverse transfection. 100 µl of the transfection
mixture was added to a final concentration of 40 and 60 nM RNA per well in a 24-well
format.After 20 min. of equilibration at room temperatureHT-22 cells were seeded out
with 33,000 cells /well. Controlswere treated with 100 µl/ml Optimem I only.
Protein extracts and immunoblots- For protein extraction and subsequent analysis,
HT-22 neurons were grown at a density of 7 x 10 4 cells per well in 24-well plates or
2 x 105 cells per well in 6-well plates. For western blot analysis, HT-22 cells were
lysed with 50–150 µl 1 : 5 diluted cell lysis reagent 5x (Promega,Mannheim,
Germany), Supplementaryed with 1 tablet per 10 ml Complete MiniProtease Inhibitor
Cocktail (Roche, Mannheim, Germany). After centrifugation at15,000 x g for 15 min.
at 4 °C, the supernatants were stored at -80°C until furtheruse. Protein amounts were
determined with the Pierce BCA kit (PerbioScience, Bonn, Germany).Absorption at
590 nm was determined using a microplate reader (Fluostar OPTIMA, BMG Labtech,
Offenburg, Germany) and protein amounts of the test samples were calculated from
the standard curve.
Western blot analysis was performed as previously described (39). Briefly, the
proteins were blotted from the gel to the membrane for 1 h at 15 V using the Trans1
Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Munich, Germany). All
primary antibodies were diluted in Tris-buffered saline with Tween 20 (TBST). The
dilution of the anti-Drp1, Tim23-antibody (BD Bioscience Laboratories, Heidelberg,
Germany) was 1:500, anti-α-tubulin-antibody (Sigma-Aldrich, Taufkirchen, Germany)
was diluted 1:10,000, AIF (Santa Cruz), CoxIV, Bid, Bcl-xl, Bax-antibodies (Cell
Signaling, Danvers, Massachusetts, USA and New England Biolabs GmbH,
Frankfurt, Germany) were diluted 1:1,000. Anti-actin antibody (MP Biomedicals,
Eschwege, Germany) were used at dilutions of 1:10,000. All secondary antibodies
were purchased from Vector Labs (Burlingame, California, USA). Horse radish
peroxidase (HRP) labeled anti-mouse IgG (H+L), anti-goat IgG (H+L) and anti-rabbit
IgG (H+L) secondary antibodies were used for western blots at dilutions of 1:2,000 1:5,000 in TBST. Equal protein loading and blotting quality was controlled by reprobing the membrane with the monoclonal anti-α-tubulin or anti-actin antibodies
(Sigma-Aldrich, Taufkirchen, Germany) and the respective secondary antibodies.
CoxIV, Tim23 and AIF were used as mitochondrial purity marker and tubulin and
actin as a purity marker for the cytosol. Chemidoc software (Bio-Rad, Munich,
Germany) was used for quantification of western blot signals.
Immunoprecipitation of Drp1 - The immunoprecipitation of Drp1 was performed by
pull-down of Drp1 from total protein lysates according to the manufacturer’s protocol
(Invitrogen, Karlsruhe, Germany). Briefly, magnetic Dynabeads Protein A were
prepared for each condition to effectively bind the Drp1-antibody (7.5 µg, BD
Biocience Laboratories, Heidelberg, Germany). The Drp1-antibody is added to the
Dynabeads Protein A for 30 min. to bind the Dynabeads via their Fc-region. For all
washing steps the tube was placed on a Dynamagnet, where the beads migrate to
the side of the tube facing the magnet and allow for easy removal of the supernatant.
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To avoid co-elution of the antibody and to increase the binding capacity of the
antibody on the Dynabeads, crosslinking reaction with BS 3 was performed according
to the manufacturer’s protocol. The bead-bound antibody is now ready for
immunoprecipitation. For immunoprecipitation of Drp1, 2.5 mg of total protein lysate
of each treatment condition was incubated for one hour at room temperature (RT)
followed by a second hour at 4 °C. The elution of Drp1 with its binding partners was
performed by adding 70 µl of 2.5x sodium dodecyl sulfate (SDS) -sample buffer and
afterwards boiling of this Dynabead-protein lysat-mix for 10 min. at 95 °C. Dynabeads
were removed from the solution and the supernatant removed. The supernatant was
stored at -80 °C. 30 µl of the eluat was loaded on a SDS-gel to detect proteininteraction partners of Drp1 by western blotting analysis. The regular protein lysate
with 30 µg of protein per treatment condition was used as a control in the SDSPAGE.
Mitochondrial fractionation-Mitochondrial fractionation was performed by the adopted
protocol from Muqit et al. 2006 with 10 million cells per treatment condition. Briefly,
cells were harvested by 1x TE and washing twice with 1x PBS. Cells were twice
disrupted by glass homogenisator containing the buffer with 250 mM Sucrose, 20 mM
Hepes, 3 mM EDTA and protease inhibitor with a pH =7.5
followed by a
centrifugation at 830 g, 10 min. at RT to producecrude nuclear pellets.The
supernatants were recovered and centrifuged at 16,800 g for 15min. yielding
cytosolic (supernatant) and mitochondrial (pellet) fractions. Protein determination of
the lysates forcytosolic, mitochondrial and nuclear fractions were prepared as
previously reported andsubjected to immunoblot analysis.
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