The following antibodies were used: anti-FLAG (Sigma, Cat: F3165, dilution 1:10,000), anti-Myc (Abcam, Ab9106, 1:2500), anti-Phosphothreonine (pThr, Thermo Fisher, 71-8200, 1:2500); anti-Phosphoserine (pSer, Thermo Fisher, 61–8100, 1:2500); anti-LETM1 (Santa Cruz, mouse sc-514136, 1:2500 or Novus Biologicals, rabbit NBP1-33433, 1:10,000). Anti-PINK1 (Abgent, mouse AM6406a, 1:5000 and Novus Biologicals, rabbit BC100–494, 1:5000), anti-Tyrosine hydroxylase (TH, EMD Millipore, AB152, 1:1000), anti-MCU (Cell Signaling, 14997, 1:10,000), anti-NCLX (Abcam, ab136975, 1:5000). Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT192RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).
Anti mcu
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-MCU is a primary antibody used for the detection of the Mitochondrial Calcium Uniporter (MCU) protein in Western blotting applications. MCU is a key component of the mitochondrial calcium uptake machinery. This antibody can be used to study the expression and localization of the MCU protein in various cell and tissue types.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab9106, by Abcam (1 mentions)
Ab152, by Cell Signaling Technology (1 mentions)
Bc100 494, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti myc, by Abcam (1 mentions)
Oxphos antibody cocktail, by Abcam (1 mentions)
Imagequant tl, by GE Healthcare (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Anti gapdh 10r g109a, by Biosynth (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Anti vinculin, by Santa Cruz Biotechnology (1 mentions)
Grp75, by Santa Cruz Biotechnology (1 mentions)
Tom20, by Santa Cruz Biotechnology (1 mentions)
Ab52175, by Abcam (1 mentions)
Ab101465, by Abcam (1 mentions)
Anti mhc, by Santa Cruz Biotechnology (1 mentions)
7 protocols using anti mcu
1
Comprehensive Antibody Profiling Protocol
2
Western Blot Analysis of Mitochondrial Proteins
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HL-1 atrial cardiomyocytes were lysed in RIPA assay buffer. Human atrial appendage tissue samples were lysed in sample buffer (15% glycerol; 1% SDS; 12,5% 0.5 M Tris, pH 6.8; 2% bromophenol-blue solution). For Western blot analysis, equal amounts of protein lysates were separated on SDS-PAGE 4–20% mini-PROTEAN TGX or 4–20% Criterion TGX precast gels (Bio-Rad, Lunteren, The Netherlands) and transferred to nitrocellulose membranes (Bio-Rad). Subsequently, membranes were incubated with primary antibodies. Signals were detected by the Amersham ECL prime Western blotting detection reagent (GE Healthcare Life Sciences, Hoevelaken, The Netherlands) utilizing the Amersham Imager 600 (GE Healthcare Life Sciences) and quantified by densitometry (ImageQuantTL, GE Healthcare Life Sciences). The following primary antibodies were used: Anti-HSP60 (ADI-SPA-805, Enzo Life Sciences, Farmingdale, NY, USA), anti-TOM20 (MCA4300Z, Bio-Rad), anti-MCU (14997S Cell Signalling Technology, Leiden, The Netherlands), OXPHOS Antibody Cocktail (MS604, Abcam) and anti-GAPDH (10R-G109a, Fitzgerald, Acton, MA, USA). Horseradish peroxidase-conjugated anti-mouse or anti-rabbit (Dako, Denmark) were used as secondary antibodies, depending on the species origin of the primary antibody.
3
Quantitative Analysis of Mitochondrial Proteins
4
Comprehensive Protein Profiling of Cell and Tumor Lysates
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Whole-cell and tumour lysates were isolated using RIPA buffer supplemented with protease inhibitors (Complete Mini, Sigma-Aldrich Inc.) and phosphatase inhibitors including sodium pyrophosphate, β-glycerophosphate, sodium fluoride and sodium orthovanadate (Sigma-Aldrich). The following primary antibodies were used: anti-MCU (#D2Z3B 1:1000, Cell Signaling), anti-MICU1 (#HPA037479 1:1000, Sigma-Aldrich), anti-MICU2 (#ab101465 1:1000, Abcam), anti-phospho-SMAD3 (#C25A9, 1:1000, Cell Signaling), anti-SMAD3 (#9513, 1:1000, Cell Signaling), anti-MYOG (#sc-12732, 1:500, Santa-Cruz), anti-MHC (#sc-32732, 1:250, Santa-Cruz) anti-HSP60 (#611563, BD Biosciences), anti-phospho-NF-κB (#3037, 1:1000, Cell Signaling), anti-NF-κB (#ab52175, 1:500, Abcam), anti-phospho-p38 MAPK (#9211, 1:1000, Cell Signaling), anti-p38 MAPK (#9212, 1:1000, Cell Signaling), and anti-β-actin (#A2228, 1:10,000, Sigma-Aldrich). Appropriate secondary antibodies (IgG-Fc Specific-Peroxidase) of mouse or rabbit origin (Sigma-Aldrich) were used.
5
Analysis of Cardiac Protein Expression
6
Mitochondrial Calcium Regulation Assay
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Culture medium (Dulbecco’s modified Eagle’s medium [DMEM]/F-12) (#11320), fetal bovine serum (FBS), insulin-transferrin-selenium (ITS), Ca2+ Green 5N, and Pierce BCA protein assay kit were from Thermo Fischer Scientific; thapsigargin, adrenaline, safranin-O, BAPTA-AM, CGP-37157, ruthenium red, UK5099, BPTES, etomoxir, and RIPA buffer were from Sigma-Aldrich; lipofectamine RNAiMAX, lipofectamine 2000, Trizol reagent, and Fura-2 AM were from Invitrogen; CMV-mito-GEM-GECO1 plasmid (#32461) was from Addgene; Bradford was from Bio-Rad Laboratories; siRNAs against MCU (ID s103465), NCLX (ID s100747), or negative control (#4390844) were from Ambion Inc; anti-MCU (#14997S) was from Cell Signaling; anti-β-actin (#ab8226) was from Abcam; fluorescent secondary antibodies (goat anti-mouse #926-68070 and goat anti-rabbit #926-68071) were from Licor.
7
Mitochondrial Protein Analysis in Mouse Tissues
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The mice were heparinized (200 U/mouse, i.p.) and sacrificed by cervical dislocation. The liver, kidney and left ventricle were quickly excised after thoracotomy. Mitochondria were isolated by a differential centrifugation method and Western blot analyses were performed as described previously [25 (link),27 (link)]. The primary antibodies used were: custom-made anti-NCLX (1:500, [25 (link)]), anti-MCU (1:1000, Cell Signaling Technology, Danvers, MA, USA, #14997), and anti-COX IV (1:2000, Abcam, Cambridge, UK, ab14744). The secondary antibodies used were: HRP-linked anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for the NCLX and MCU, and HRP-linked anti-mouse IgG (GE healthcare, Chicago, IL, USA) for the COX IV. The quality of the custom-made anti-NCLX antibody, which was verified using HeLa cells transiently expressing NCLX and NCLX–FLAG [25 (link)], was further confirmed in Western blot analysis using cardiac mitochondria, where a single positive band around 65 kDa was diminished by pre-incubating the antibody with an excess amount of antigen peptide (Figure S1 ).
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