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6 protocols using mouse anti mtco1

1

Mitochondrial Dynamics Regulation Assay

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Chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany) except Mdivi-1 (Enzo Life Science, Lörrach, Germany) as well as doxycycline, blasticidin and zeocin (Invivogen, San Diego, CA, USA). Cell culture materials and media were obtained from Biochrom (Berlin, Germany). MitoTracker GreenFM (MTG) and MitoSOX™ Red superoxide indicator (MitoSOX) were purchased from Molecular Probes (Eugene, USA).
The following primary antibodies were used: rabbit anti-DNP (Sigma-Aldrich, Deisenhofen, Germany), rabbit anti-PINK1 (Cell Signaling, Boston, MA, USA), mouse anti-β-actin (Cell Signaling, Boston, USA), rabbit anti-COX IV (Cell Signaling, Boston, MA, USA), rabbit anti-Lon protease (Abcam, Cambridge, UK), mouse anti-GAPDH (Abcam, Cambridge, UK), rabbit anti-Ki-67 (Abcam, Cambridge, UK), mouse anti-CDKN2A/p16INK4α (Abcam, Cambridge, UK), rabbit anti-p21 Waf1/Cip1 (Cell Signaling, Boston, MA, USA), mouse anti-MT-CO1 (Abcam, Cambridge, UK), mouse anti-SDHA (Abcam, Cambridge, UK), mouse anti-VDAC (Abcam, Cambridge, UK) and rabbit anti-Fis1 (Cell Signaling, Boston, MA, USA). Secondary antibodies used for immunoblotting were purchased from LI-COR Biosciences (Lincoln, AL, USA). The FITC-labeled antibody for immunofluorescence was purchased from Invitrogen (Carlsbad, CA, USA).
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2

Antibody Characterization for EGFR Signaling

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The primary antibodies used for immunoblot analysis and immunohistochemical staining were rabbit anti-EGFR, mouse anti-vinculin, mouse anti-Tom23, goat anti-Tim23, rabbit anti-Mfn1, mouse anti-Drp1 (all above from Santa Cruz Biotechnology, CA, USA), mouse anti-MTCO1 (Abcam, Cambridge, UK), rabbit anti-phospho-EGFR (Tyr1068), rabbit anti- phospho-Drp1 (Ser637) (all above from Cell Signaling Technology, Inc., MA, USA), mouse anti-OPA1 (BD Biosciences, NJ, USA) and mouse anti-myc, mouse anti-Flag, mouse anti-β actin antibody (all above from Sigma-Aldrich, Inc., MO, USA). The primary antibodies used for immnofluorescence staining were rabbit anti-EGFR (Abcam, Cambridge, UK) antibody.
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3

Western Blot Analysis of Mitochondrial Proteins

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Western blot analysis was performed as previously described [36 (link)]. Primary antibodies were diluted in TBS/0.1% Tween 20 (TBS-T) and incubated overnight at 4°C and were Drosophila anti-TFAM (Abcam, 1/500), mouse anti-ATP5A (Abcam, 1/5000), mouse anti-MTCO1 (Abcam, 1/1000) and rabbit anti-Actin (Cell Signaling, 1/4000). After three ten minute washes in TBS-T, the membranes were incubated for 90 minutes with fluorescently labelled secondary antibodies (anti-mouse IRdye 680 and anti-rabbit IRdye 800, LI-COR, both at 1/5000) diluted in TBS-T, then washed three times for ten minutes in TBS-T. The membranes were then scanned and analysed using an Odyssey infrared scanner (LI-COR). Odyssey infrared imaging systems application software version 3.0.25 was used to quantify the intensity of the bands on the blots. Normalised expression level was calculated by determining the band intensity relative to Actin.
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4

Quantification of Mitochondrial Proteins

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Purified lung epithelial cells were lysed in 2x Western blotting buffer (120 mM Tris-Cl pH 6.8, 4% SDS, 20% Glycerol, 200 mM DTT, 0.02% Bromophenol Blue). The lysates were centrifuged at 16,100 x g for 15 min at 4 °C before loading onto SDS-PAGE. The primary antibodies used were rabbit anti-Raptor (1:2000, EMD Millipore Corporation, Cat# 09-217; RRID:AB_1659713), mouse anti-S6 ribosomal protein (1:2000, Cell Signaling Technology, Cat# 2317; RRID:AB_2238583), rabbit anti-phospho-S6 ribosomal protein (Ser235/236) (1:2000, Cell Signaling Technology, Cat# 4856), rabbit anti-MLC2 (1:500; Cell Signaling Technology, Cat#3672; RRID:AB_10692513), goat anti-TFAM (1: 250, Santa Cruz Biotechnology, Cat# sc-23588; RRID:AB_2303230), rabbit anti-COX10 (1:500, Proteintech, Cat# 10611-2-AP; RRID:AB_2084833), mouse anti-MTCO1 (1:500, Abcam, Cat# ab14705; RRID:AB_2084810), and mouse anti-alpha-tubulin (1:3000, Developmental Studies Hybridoma Bank, Cat# 12G10; RRID:AB_1157911). Western blotting images were captured on a LI-COR Odyssey Fc device. The uncropped images are available in the Source Data file.
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5

Quantifying Mitochondrial Protein Levels

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Protein samples were separated by polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore). Blots were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h and then incubated with primary antibodies. The following antibodies were used: mouse anti-MTCO1 (1:2,000, Abcam, Cambridge, UK), mouse anti-complex IV subunit (COX IV; 1:2,000, Abcam), mouse anti-ATPB (1:2,000, Abcam), Total Oxidative Phosphorylation (OXPHOS) Human WB Antibody Cocktail (1:200, Abcam), mouse anti-VDAC1/Porin (1:1,000, Abcam). Peroxidase-conjugated goat antimouse IgG (1:10,000, Bio-Rad) and antirabbit IgG (1:5,000, Bio-Rad) were used as secondary antibodies. To control for the amount of protein loaded, we used a mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-GAPDH-loading control antibody (1:10,000, Abcam). Immunoreactive proteins were visualized using an enhanced chemiluminescence detection system (Clarity ECL, Bio-Rad), and bands were detected with an ImageQuant LAS 4000 Imaging System (GE Healthcare Life Sciences, Barcelona, Spain).
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6

Comprehensive Antibody Panel for OXPHOS

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The following antibodies were used in our work: rabbit anti-NDUFB8 (Proteintech, 1479-1-AP); mouse anti-MTCO1 (Abcam, ab14705); mouse anti-total OXPHOS antibody cocktail (Abcam, ab110413); mouse anti-GFP (Roche, 11814460001); rabbit anti-VPS39 (Proteintech, 16219-1-AP and Novus Biologicals, NBP1-76535); mouse anti-KDEL (Millipore, 10C3); mouse anti-TOM40 (Santa Cruz, sc-365467); rabbit anti-TOM20 (Proteintech, 11802-1-AP); rabbit anti-LC3B (Sigma, L7543); rabbit anti-MICU1 (Sigma, PA5-83371); guinea pig anti-p62 (Progen, GP62-C); rabbit anti-pS473-AKT (Cell Signaling, 4060S), rabbit anti-AKT (Cell Signaling, 4685); rabbit anti-pT246-PRAS40 (Cell Signaling, 13175); anti-PRAS40 (Cell Signaling, 2691); rabbit anti-GAPDH (Santa Cruz, sc-25778); mouse anti-actin (Sigma, A5316 and abcam, ab14128); mouse anti-tubulin (Sigma, T6074); rabbit anti-SGPL1 (Atlas Antibodies, HPA021125); rabbit anti-AIF (Cell Signaling, 5318).
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