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5 protocols using anti atpb

1

Coimmunoprecipitation of Protein Complexes

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Cells were lysed at 4 °C in extraction buffer (0.1 M NaCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 5 mM NaF, 1 mM dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail and phosphatase inhibitors). Homogenates were cleared by centrifugation (12,000 g, 10 min). Coimmunoprecipitation was performed according to previous techniques55 (link). In vitro IP was performed using purified recombinant proteins instead of the cell lysate. The antibodies used in the study were as follows: Anti-Myc (Cell Signaling, #2276); Anti-DDK (Origene, TA50011); Anti-DJ-1(Santa Cruz, sc27006 and sc32874; Cell Signaling, 2134S); Anti-β-Actin, #4970); Anti-GAPDH (Santa Cruz, sc-32233); Anti-mATP5G1/2/3(abcam, ab180149); Anti-ATPB (abcam, ab14730), and Anti-Bcl-xL(Cell Signaling, #2764); Anti-Puromycin (3RH11) (Kerafast, EQ0001).
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2

Immunostaining of Primary Hippocampal Neurons

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Primary hippocampal neurons fixed in 10% buffered formalin were blocked in 10% goat serum for 1 h, then incubated with anti-HA (1:10 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GFP (1:100, 1:100, Abcam), anti-ΔN-Bcl-xL (1:10 dilution, Aves Labs), and anti-ATPB (1:100 dilution, Abcam) overnight at 4 °C. Cells were washed and incubated with Alexa-fluor 488 antibody or Alexa-568 antibody (1:200 dilution, Invitrogen, Molecular Probes, Carlsbad, CA, USA) for 1 h at room temperature and mounted on glass slides. Images were taken with a Zeiss Axiovert 200 microscope (Zeiss, Oberkochen, Germany) and processed using AxioVision 4.8.
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3

Immunofluorescence Analysis of ATPB, Drp1, and p62

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BMDMs were washed twice, fixed by 4% cold paraformaldehyde for 30 min, permeabilized by 0.5% Triton X‐100 for 10 min, blocked by goat serum for 1 h, and incubated with anti‐ATPB (1:100, Abcam) overnight at 4°C. On the second day, BMDMs were stained with Goat anti‐mouse IgG H&L (Alexa Flour® 488, Abcam) for 1 h, followed by a second block and primary incubation (anti‐dynamic‐related protein 1 (anti‐Drp1), 1:250, Abcam; anti‐SQSTM1/p62, 1 μg/mL, Abcam). On the third day, a secondary antibody (Alexa Flour® 555, Abcam) was added to incubate with cells for 1 h. DAPI was used to stain the nucleus. Slides and stained cells were observed and photographed by a confocal microscope.
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4

Antibodies for Cell Cycle Analysis

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Rabbit polyclonal affinity–purified anti-Cyclin Ba/b (acetyl-NRDLNIQESGPVKAVVNAC-amide; acetyl-CLEFLRRFSRVAEETIDPKEY-amide), anti-CDK1b (acety1-CPDFTKPSTLNVNTSLNEMDM-amide), anti-CDK1c (acetyl-MRKRNIDRPPTQDLNNYC–amide), ant-CDK1d (acety1-MIKAKSGTQDLSNYKRC-amide), and anti-lamin1 (acetyl-QSPISLPPLSGSTC-amide) were from 21st Century Biochemicals (Marlboro, MA). Other antibodies: anti-PSTAIR (Abcam, ab10345), anti-ATPB (Abcam, ab37922), anti-histone 3 (Abcam, ab10799), anti-H3-S28 (Abcam, ab10543), anti-GFP (AMS biotechnology, M30939-2). Secondary antibodies for immunofluorescence: goat anti-rabbit IgG (H+L) Alexa Fluor 568 (Thermofisher, A-11011); and for western blots: goat anti-rabbit IgG (H+L) superclonal™ secondary antibody, HRP (Thermofisher, A27033), Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch, 115-035-003).
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5

Immunostaining of Stem Cells

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For immunostaining ESCs and EpiSCs were fixed for 10min in 4%PFA at room temperature, permeabilised in 0,4% Triton-X100/PBS for 5 minutes at room temperature, blocked in 10%BSA/ 0,1% Triton X-100/PBS and incubated overnight at 4°C in primary antibody diluted in 1%BSA/0,1%Triton X-100 (anti-Cleaved Caspase 3 Asp175, Cell Signalling, 1:100; anti-NANOG (eBioscience, 1:100), anti ATP-b (Abcam, 1:200), anti-P62 (BD, 1:100). Alexa-Fluor conjugated secondary antibodies (Thermo Fisher Scientific, 1:600) were used at 1/500 dilution in 1%BSA/0,1%Triton X-100. Cells were mounted for visualization in Vectashield with DAPI (Vector Laboratories). Images were acquired with a Zeiss confocal microscope and analysed with the Fiji software (Schindelin et al., 2012 (link)).
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