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5 protocols using anti mfn1

1

Antibody Profiling of Metabolic Regulators

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The primary antibodies used against the proteins were as follows: Anti-actin (1:1,000), obtained from PumeiBiotechnology (Pumei, China); anti-fibronectin (1:1,000), anti-collagen-I (1:1,000), anti-α-smooth muscle actin (α-SMA, 1:1000), anti-interleukin 6 (IL-6,1:1000), anti-PPARα (1:1000 for Western blot, 1:100 for immunohistochemical staining), anti-CPT1a (1:1,000), anti-Mfn1 ((1:1,000 for Western blot, 1:100 for immunohistochemical staining) and anti-Drp1, which were obtained from ProteintechGroup (Proteintech, China);
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2

Mitochondrial Dysfunction and Cell Death

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TMZ, Compound C, AICAR, Mdivi1, MG132 and WY14643, were purchased from MedChemExpress company. MTT and N‐Acetyl‐L‐cysteine (NAC) were purchased from Sigma‐Aldrich. MitoTracker™ Red FM (M22425), Hoechst 33342 (H1399) and anti‐Ubiquitin WB Antibody (13–1600) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) (1:1,000). Anti‐phospho‐Ubiquitin (Ser65) (ABS1513‐I) was purchased from Merck KGaA company (1:1000). Anti‐P53 (21891–1‐AP), anti‐PINK1 (23274–1‐AP), anti‐Parkin (14060–1‐AP), anti‐Drp1 (12957–1‐AP), anti‐Opa1 (27733–1‐AP), anti‐Mfn1 (13798–1‐AP), anti‐Mfn2 (12186–1‐AP), anti‐Caspase‐9 (10380–1‐AP), anti‐BAX (50599–2‐Ig), anti‐Caspase‐3 (19677–1‐AP), anti‐VDAC1 (10866–1‐AP), anti‐Lamin B (12987–1‐AP), anti‐Cytochrome c (10993–1‐AP), and anti‐β‐actin (60008–1‐Ig) were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti‐γ‐H2A.X (ab81299) was purchased from Abcam (1:1000). Anti‐AMPKα (2532) and p‐AMPKα (50081) were purchased from Cell Signaling Technology, Inc. (Massachusetts, USA) (1:1000). Anti‐phospho‐DRP1(Ser616) (DF2972) was purchased from Affinity Biosciences (1:1000). Anti‐phospho‐DRP1(Ser637) (6319S) was purchased from Cell Signaling Technology, Inc. (1:1,000).
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3

Mitochondrial Dynamics and Cell Signaling

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NaHS was purchased from Sigma-Aldrich (St Louis, MO, USA), and the cigarettes were purchased from Guangdong Tobacco Industry Co., Ltd. (Guangzhou, China). SRT1720 and EX 527 were purchased from Selleck Chemicals (Houston, TX, USA). The TRIzol Reagent was purchased from Ambion (Life Technologies, CA, USA). The PrimeScript RT reagent Kit with gDNA Eraser was from Takara Bio Inc. (Takara, Shiga, Japan), and the SsoFast EvaGreen Supermix was obtained from Bio-Rad Laboratories, Inc. (CA, USA). The primary and second antibodies described in this study include: anti-Bcl-2 and anti-β-actin polyclonal antibodies were purchased from Proteintech (Chicago, IL, USA); anti-MFN1, anti-SIRT1, anti-FOXO3 and anti-Bax antibodies were purchased from Abclonal (Wuhan, China); anti-p21 and anti-p53 antibodies were purchased from Cell Signaling Technology (CA, USA); anti-FIS1 antibodies, and the horseradish peroxidase (HRP)-labeled Goat Anti-Rabbit/Mouse IgG (H+L) were purchased from Abcam Biotechnology (Cambridge, MA, USA). The poly-vinylidene fluoride (PVDF) membranes were from Millipore Corporation (Billerica, MA, USA). ECL-Plus detection kit probed was purchased from Tanon Science and Technology Co., Ltd. (Shanghai, China). Other reagents were all purchased from GBCBIO Technologies Inc. (Guangzhou, China) unless otherwise indicated.
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4

Mitochondrial Dynamics Protein Detection

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Western blot was performed as previously described [52 (link), 53 (link)]. The following primary antibodies were used for WB detection: anti-MFN1 (1 : 500), anti-OPA1 (1 : 1000), and anti-DRP1 antibodies (1 : 1000) were all purchased from Proteintech (Chicago, IL, USA); and anti-Bax (1 : 2000), anti-Bcl-2 (1 : 2000), anti-caspase-3 (1 : 2000), and anti-GAPDH antibodies (1 : 10000) were all purchased from Abcam (Cambridge, Cambs, UK). GAPDH served as internal reference. WB bands were detected with Gel-Pro Analyzer (Media Cybernetics, MD, USA).
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5

Western Blot Analysis of Mitochondrial Dynamics

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Protein extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes as previously described [23 (link)]. The membranes were incubated with the following antibodies: anti-VEGFA (Proteintech, 66828-1-1g); anti-pERK1/2 (Cell Signaling, 4370), anti-ERK1/2 (Cell Signaling, 4695), anti-MFN1 (Proteintech, 13798-1-AP), anti-MFN2 (Proteintech, 12186-1-AP), anti-pDRP1 ser616 (Cell Signaling, 3455), anti-DRP1 (Proteintech, 12957-1-AP), and anti--actin (Cell Signaling, 3700), at 4C overnight. After washing, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies at 37C for 1h. The immunoreactive bands were visualized with enhanced chemiluminescence reagent (Thermo Fisher, 32109) and imaged with the ChemiDoc XRS Plus luminescent image analyzer (Bio-Rad). Densitometric quantification of band intensity from 4 independent experiments was conducted with Image-Pro Plus 6.0 software.
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