For western blotting, cell extracts were prepared by addition of ice cold RIPA buffer (300 μL per well of a 12‐well plate; 1 mL per T25 or T75 cell culture flask) containing protease inhibitor cocktail (200 μL·40 mL−1). Cells were scraped from the adherent layer, the cell suspension was centrifuged at 12000 g for 15–20 min at 4°C and the supernatant analysed for protein before western blot analysis was performed as described above using primary antibodies detecting Nox2 (1:1000, Abcam ab129068) and Mfn2 (1:1000 Abcam ab50838). For measurement of ROS production, homogenates were prepared by probe sonication of the whole cell preparation on ice for 20 s and lucigenin‐enhanced chemiluminescence determined as above.
Ab50838
Manufactured by Abcam
Sourced in United Kingdom
Ab50838 is a lab equipment product offered by Abcam. It serves as a general-purpose laboratory tool. The core function of this product is to perform a specific task within the laboratory setting. No further details about the intended use or capabilities of this product can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab56788, by Abcam (2 mentions)
Ab131607, by Abcam (2 mentions)
Ab57602, by Abcam (2 mentions)
Ab126575, by Abcam (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Ab129068, by Abcam (1 mentions)
Ab14744, by Abcam (1 mentions)
Ab71498, by Abcam (1 mentions)
Ab54481, by Abcam (1 mentions)
Ab90857, by Abcam (1 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Ab2769, by Abcam (1 mentions)
Sodium dodecyl sulfate (sds), by Beyotime (1 mentions)
Sc 514544x, by Santa Cruz Biotechnology (1 mentions)
Ab110413, by Abcam (1 mentions)
Cpt 1, by Santa Cruz Biotechnology (1 mentions)
Mic19, by Proteintech (1 mentions)
Tom20, by Cell Signaling Technology (1 mentions)
Mic60, by Proteintech (1 mentions)
β tubulin, by Merck Group (1 mentions)
9 protocols using ab50838
1
Western Blotting and ROS Measurement
2
Comprehensive Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using a cell lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS (Beyotime, Shanghai, China) supplemented with each protease and phosphatase inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany) per 10 ml solution. Western blot was performed as previously described 57 (link). Primary antibodies against IRF1 (sc-514544x) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies against P62 (5114), p-mTOR (5536), t-mTOR (2983), p-p70S6K (9234), p-4EBP1 (2855), PINK1 (6946) and Parkin (4211) were obtained from Cell Signaling Technology (CST, Danvers, MA, USA). The antibodies against DRP1 (ab56788), MFN1 (ab57602), MFN2 (ab50838), OPA1 (ab90857), FIS1 (ab71498), AhR (ab2769), COXIV (ab14744), PGC1α (ab54481) and TFAM (ab131607) were purchased from Abcam (Cambridge, MA, USA). LC3 antibody (L7543) was purchased from Sigma-Aldrich.
3
Protein Extraction and Analysis from Skeletal Muscle
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen SkM was thawed and immediately homogenized in RIPA lysis buffer (Tris-HCl 10 mM, pH 7.4; EDTA 5mM; NaCl 50 mM; sodium deoxycholate 0.1%; triton X-100 1% v/v) supplemented with protease and phosphatase inhibitors (Roche Applied Sciences, Germany) using a motorized pestle system (Pellet Pestle Cordless Drive Unit, DKw Life Sciences, USA). The total lysate was centrifuged at 12,000 rpm three times for 10 min at 4 °C; the supernatant was collected for protein determination using BCA assay (Novagen, Merck Ma, NJ, USA). Protein separation and immunoblotting were performed, as previously described [42 (link)]. Immunoblotting was performed using the following antibodies: Mfn2 (ab50838, Abcam, 1:1000), Mfn1 (ab126575, Abcam, 1:1000), Opa1 (ab42634, Abcam, 1:1000), Drp1 (PA1-16987, Thermo, 1:1000), phosphoserine 616-Drp (5616, Cell Signaling, 1:1000), Total OXPHOS cocktail (ab110413, Abcam, 1:1000), Mic60 (#10179-1-AP, Proteintech, 1:1000), Mic19 (#25625-1-AP, Proteintech, 1:1000), mHsp70 (#MA3-028, Pierce, 1:1000), Oxct1 (#12175-1-AP, Proteintech, 1:1000), Acca2 (#100847, Santa Cruz, 1:1000), Scad (#365953, Santa Cruz, 1:1000), Vlcad (#376239, Santa Cruz, 1:1000), Cpt1 (#393070, Santa Cruz 1:1000), Tom20 (72610, Cell Signaling, 1:1000), and β-Tubulin (T0198, Sigma Chemical Co, 1:5000). Samples of the same muscle were run on the same gels.
4
Quantifying Mitochondrial Dynamics Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissue samples were homogenized in ice‐cold buffer (CelLytic™ MT Cell Lysis Reagent) containing protease and phosphatase inhibitor cocktails (Nacalai Tesque, Inc., Kyoto, Japan). The homogenate was centrifuged at 15,000 g for 15 min at 4°C to obtain the supernatant. Equal amounts of protein were analyzed by immunoblot assays using mouse monoclonal anti‐Mfn1 (Abcam, ab57602, 1:1000), rabbit polyclonal anti‐Mfn2 (Abcam, ab50838, 1:1000), mouse monoclonal anti‐OPA1 (BD Biosciences, 612606, 1:1000), rabbit polyclonal anti‐Fis1 (GeneTex, GTX111010, 1:1000), rabbit monoclonal anti‐phospho‐Ser616‐Drp1 (Cell Signaling Technology, #4494, 1:1000), rabbit monoclonal anti‐phospho‐Ser637‐Drp1 (Cell Signaling Technology, #6319, 1:1000), mouse monoclonal anti‐Drp1 (BD Biosciences, 611112, 1:1000), mouse monoclonal anti‐Bnip3 (Sigma Aldrich, B7931, 1:1000), and mouse monoclonal anti‐vinculin (Sigma Aldrich, V9131, 1:5000). Intensities of individual bands were quantified by using Image J software.
5
Immunohistochemical Analysis of Synaptic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This procedure was performed by a standard indirect method as described previously [28 (link)]. Primary antibodies and dilutions were as follows: anti-COX IV (1:250; # ab16056, Abcam), anti-DRP1 (1:250; # ab56788, Abcam), anti-Mfn2 (1:250; # ab50838, Abcam), anti-proBDNF (1:250; # ab72440, Abcam), anti-mBDNF (1:250; # GF35L, Millipore), anti-TrkB (1:250; # ab18987, Abcam), anti-phospho-TrkB (phospho TrkB Y817; 1:250; # ab81288, Abcam), anti-p75NTR (1:250; # ab93934, Abcam), anti-synapsin I (1:250; # ab64581, Abcam), anti-PSD-95 (1:250; # ab12093, Abcam), and anti-amyloid beta peptide (MOAB-2; 1:250; # MABN254, Millipore). After incubation with the respective secondary antibodies DyLight-650 or Alexa Fluor 488 (## ab96886, ab150073, Abcam) diluted 1:250, the slices were coverslipped with the Fluoro-shield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI; # ab104139, Abcam) and examined under an Axioplan 2 microscope (Zeiss).
6
Analysis of Lipid Droplet Proteins by Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell protein extracts were obtained using T-PER lysis buffer (78510, Thermo Fisher Scientific), supplemented with protease (#04693159001, Roche) and phosphatase (#04906845001, Roche) inhibitors. Homogenates were centrifuged at 14,500 G for 10 min, and the supernatants were collected for measuring protein concentrations with the BCA protein assay (23227, Thermo Fisher Scientific). Protein extracts of 50 μg were resolved in 10% SDS-polyacrylamide gels, transferred onto PVDF membranes previously activated with methanol, and blotted overnight with primary antibodies at 4 °C. The antibodies and dilutions were: anti-PLIN2 (1:1000; #MA5-24797, Thermo Fisher Scientific), anti-PLIN5 (1:1000; #GP31, Progen), and anti-MFN2 (1:1000; #ab50838, Abcam). The membranes were then incubated for 1 h with the following secondary HRP-coupled antibodies: anti-mouse (1:5000; #402335, Calbiochem), anti-guinea pig (1:5000; #ab6908, Abcam) or anti-rabbit (1:5000; #401315, Calbiochem) and revealed with the EZ-ECL detection kit (DW1029, Biological Industries). A C-DiGit® Blot Scanner and the Image Studio software version 3.1 (LI-COR) were used for image acquisition and densitometric analysis. GAPDH (1:5000; #MAB374, Sigma-Aldrich) was used as a loading control.
7
Western Blotting and Immunohistochemistry for Mitochondrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blotting the following antibodies were used: anti-DRP1 (dynamin-1-like protein, BD 611112) 1:1000 and anti-OPA1 (optic atrophy protein 1, BD 612606) 1:1000 from BD Biosciences. Anti-MFN1 (anti-mitofusin1, ab126575) 1:500, anti-MFN2 (mitofusin-2, ab50838) 1:1000; anti-NDUFA9 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, ab14713) 1:3000; anti-SDHA (succinate dehydrogenase [ubiquinone] flavoprotein subunit, ab14715) 1:10000; anti-UQCRC2 (cytochrome b-c 1 complex subunit 2, ab14745) 1:6000; anti-MTCO1 (cytochrome c oxidase subunit 1, ab14705) 1:6000; anti-ATP5A (ab14748) 1:6000 and anti-citrate synthase (ab129095) 1:1000 from Abcam. For detection of the phosphorylation state anti-Phospho-DRP1 (Ser616) (3455, Cell Signaling) 1:1000 was used. For analysis of HTT aggregates the S829 (1:3000) antibody, kindly provided by Gillian Bates (UCL, UK), was used. As secondary antibodies goat anti-mouse IgG (H+L)-HRP (1:3000, 1706516, Bio-Rad), goat anti-rabbit IgG (H+L)-HRP (1:10000, 111-035-003, Jackson ImmunoResearch) or rabbit anti-goat IgG (H+L)-HRP (1:10000, R21459, Life Technologies) were used.
For immunohistochemistry the S829 antibody (1:400 in PBS, 5% horse serum, 0.05% Tween) and as secondary antibody donkey anti-sheep IgG (H+L) Alexa fluor 488 (1:400 in PBS, A11015, Thermo Fisher) were used.
For immunohistochemistry the S829 antibody (1:400 in PBS, 5% horse serum, 0.05% Tween) and as secondary antibody donkey anti-sheep IgG (H+L) Alexa fluor 488 (1:400 in PBS, A11015, Thermo Fisher) were used.
8
Mitochondrial Function Regulation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell Counting Kit-8 (CCk8; CK04) was purchased from Dojindo Laboratories (Kumamoto, Japan). Glass-bottom cell culture dishes (801001) were purchased from Nest Biotechnology (Wuxi, China). DM (mast-120131108, HPLC ≥98%) was purchased from MUST Biotechnology Co., Ltd. (Chengdu, China). Dex (D4902), Dex (D1756), Akti (A6730), rapamycin (RAPA; R0395) and the Protein Carbonyl Content Assay Kit (MAK094) were purchased from Sigma-Aldrich (St. Louis, MO, USA). MitoTracker Deep Red (M22426) and MitoSOX Red (M36008) were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against myosin heavy chain (MHC; ab 24642), PGC-1α (ab 106814), TFAM (ab 131607), OPA1 (ab 157457), Tom20 (ab 56783), mfn1 (ab 104585), mfn2 (ab 50838), FoxO3a (ab 23683) and VDAC1 (ab 15895) were obtained from Abcam (Cambridge, UK). Antibodies against p-Akt (9271), Akt (9272), p-mTOR (2971), mTOR (2972), p-S6K (9205), S6K (9202) and GAPDH (5174) were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against atrogin-1 (sc-33782) and MuRF1 (sc-27642) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), whereas the antibody against drp1 (BD611113) was obtained from BD Biosciences (San Jose, CA).
9
Proteomic Analysis of Adipose Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen fat tissue (300 mg) was solubilized in 500 µl 2.5X Laemmli buffer containing 150 mM dithiotreitol. Lysates were subjected to SDS-PAGE, blotted onto nitrocellulose membranes, and probed with antibodies directed against MFN2 (1/1000; Abcam, ab50838, Cambridge, UK), SREBP-1 (1/500; SC-366, Santa Cruz Biotechnology, TX, USA), PPARSC-7196 adiponectin (1/1000; MAI-054, Affinity BioReagents, Golden United States), CITED1 (1/1000; Novus H0000443S-M03, Bio-Techne, Lille, France), FGF21 (1/1000; Abcam ab171941), PGC-1 (1/500; SC-5816), and tubulin (T5168, Sigma-Aldrich, St. Quentin Fallavier, France). Tubulin was used as a loading control.
18 F-FDG-PET scan 18 F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) with computed tomography (CT) was performed according to the EANM guidelines [27] . Patients fasted for at least 4.5 h. A Gemini Dual PET/CT camera (Philips) with time of flight (TOF) technology was used for imaging. Low-dose CT (120 kVp, 30-50 mAs) was acquired first, followed by PET acquisition 60-70 min after a weight-adjusted dose of 2.5 MBq/kilogram 18 F-FDG injection, covering a complete whole-body field of view, from the skull to the feet.
Standardized uptakes values (SUV) were calculated for the regions of interest. FDG uptake in the liver was taken as a reference.
18 F-FDG-PET scan 18 F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET) with computed tomography (CT) was performed according to the EANM guidelines [27] . Patients fasted for at least 4.5 h. A Gemini Dual PET/CT camera (Philips) with time of flight (TOF) technology was used for imaging. Low-dose CT (120 kVp, 30-50 mAs) was acquired first, followed by PET acquisition 60-70 min after a weight-adjusted dose of 2.5 MBq/kilogram 18 F-FDG injection, covering a complete whole-body field of view, from the skull to the feet.
Standardized uptakes values (SUV) were calculated for the regions of interest. FDG uptake in the liver was taken as a reference.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!