Baicalein (≥ 99%, Yousi Scientific Co., Ltd, Shanghai, China) was dissolved in DMSO at concentration of 200 mM and stored at -20 ℃. Mdivi-1, an inhibitor of Drp1, was purchased from Selleck (Huston, TX, USA). 3-Methyladenine (3-MA), an inhibitor of autophagosomes, and Bafilomycin A1 (Baf-A1), an inhibitor of H+-ATPase, were purchased from Selleck. Antibodies against PARP (#9542), Drp1 (#5391), AMPKα (#5831), p-AMPKα (Thr172) (#2535), LC3 (#12741), Bak (#6947) and β-actin (#3700) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against Caspase 3 (#19677-1-AP), Caspase 9 (#10380-1-AP), Bcl2 (#12789-1-AP), Bcl-xl (#10783-1-AP), Bax (#50599-1-AP), Cytochrome c (#10993-1-AP), Aif (#17984-1-AP), Cox IV (#11242-1-AP), Fis1 (#10956-1-AP), Opa1 (#27733-1-AP), Mfn1 (#13798-1-AP), Ndufs1 (#12444-1-AP), Sdha (#14865-1-AP), Uqcrc1 (#21705-1-AP), Atp5a1 (#14676-1-AP), p62 (#18420-1-AP), and Beclin1 (#11306-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against p-Drp1 (Ser616) (#12749) was obtained from Signalway Antibody (College Park, MD, USA). Secondary goat anti-rabbit or rabbit anti-mouse antibodies were purchased from Proteintech. Fluorescent-labeled antibody Annexin V-FITC, Annexin V-APC, PI, 7-AAD and 10 × binding buffer were obtained from BD (Franklin Lakes, New Jersey, USA).
Ndufs1
Manufactured by Proteintech
Sourced in United States
NDUFS1 is a subunit of the mitochondrial respiratory complex I. It plays a critical role in the electron transport chain and is essential for cellular respiration and energy production.
Automatically generated - may contain errors
Lab products found in correlation
Uqcrc1, by Proteintech (3 mentions)
Ndufv1, by Proteintech (2 mentions)
Uqcrfs1, by Proteintech (2 mentions)
Annexin 5 fitc, by BD (1 mentions)
Annexin 5 apc, by BD (1 mentions)
P ampkα thr172, by Cell Signaling Technology (1 mentions)
7 aad, by BD (1 mentions)
Cox 4, by Proteintech (1 mentions)
Atp5a1, by Proteintech (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cytochrome c, by Proteintech (1 mentions)
Mdivi 1, by Selleck Chemicals (1 mentions)
Bafilomycin a1 baf a1, by Selleck Chemicals (1 mentions)
3 methyladenine 3 ma, by Selleck Chemicals (1 mentions)
Bcl xl, by Proteintech (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Beclin 1, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Uqcrc2, by Abcam (1 mentions)
Ndufs2, by ABclonal (1 mentions)
8 protocols using ndufs1
1
Baicalein Inhibits Mitochondrial Dynamics
2
Immunoblot Analysis of Mitochondrial Proteins
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Here, 20 µg of total cellular proteins obtained from MSCs were denatured and loaded on sodium dodecyl sulfate polyacrylamide gels. After electrophoresis, the gels were transferred to a PVDF membrane (Millipore) and processed for immunoblotting. Commercially available antibodies, such as MTU1 (ab50895), ND5 (ab92624), NDUFB8 (ab110242), SDHB (ab14714), UQCRC2 (ab14745), ATP5a (ab14748), and MTCO1 (ab17405) from Abcam, ATP6 (55313-1-AP), GAPDH (60004-1-Ig), and VDAC (55259-1-AP) from Proteintech, NDUFS1 (A16926), NDUFS2 (A12858), ND4 (A17970), and CYTB (A17966) from Abclonal were used. Peroxidase AffiniPure Goat Anti-Mouse IgG and Goat Anti-Rabbit IgG (Jackson) were used as secondary antibodies, and the protein signals were detected using the ECL system. Band intensities were quantified from the 16-bit digital image by densitometry in ImageJ and are shown normalized to GAPDH for each target.
3
Western Blot Analysis of Protein Markers
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Western blot was carried out as described previously [32 ]. The specific primary antibodies against PHB2 (1:2000), β-actin (1:5000), Flag tag (1:1000), NDUFS1 (1:2000), HA tag (1:4000), GST tag (1:5000), and NDUFV1 (1:1000) were purchased from Proteintech Group. Immunoreactive bands were revealed with the ECL Advance Western Blotting Detection Kit (Amersham Bioscience) and visualized by Tanon 4500SF image system (Tanon, Shanghai, China).
4
Antibody Purchasing and Handling Protocol
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The DATS (purity about 99%) was purchased from LKT Laboratories (St. Paul, MN, USA), dissolved in dimethyl sulfoxide (DMSO; 28 mM stock), and stored at –80°C prior to use. Antibodies against neurofascin (NFASC), ribosomal protein L11 (RPL11), ribosomal protein S14 (RPS14), NADH:ubiquinone oxidoreductase core subunit V1 (NDUFV1), and NADH:ubiquinone oxidoreductase core subunit S1 (NDUFS1) were purchased from Proteintech Group (Rosemont, IL, USA). The N-acetyltransferase domain containing 1 (NATD1) antibody was from Aviva Systems Biology (San Diego, CA, USA). The antibodies against p38 α/β mitogen-activated protein kinase (MAPK), phospho-(Tyr 182)-p38, extracellular-signal-related kinase 1 (ERK1), and phospho-(Tyr 204)-ERK were from Santa Cruz Biotechnology (Dallas, TX, USA). The c-Jun N-terminal kinase 2 (JNK2) antibody not recognizing JNK1 or JNK3 but detecting endogenous JNK2 at a molecular weight of 46 and 54 kDa and phospho-(Thr183/Tyr185)-JNK antibody were from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from GeneTex (Irvine, CA, USA).
5
Protein Extraction from Transfected Cells
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For protein extraction, transfected HEK293 cells with hMfsd7c, mMfsd7c, hMfsd7c mutants were lysed with RIPA buffer, respectively. For validation of deletion of Mfsd7c in mice, micro-vessels from PBS-perfused brains of WT and EcMfsd7c-KO mice were isolated, homogenized and lyzed with RIPA buffer. For Western blot analysis of hMfsd7c expression in yeasts, 1 mL of yeast culture at OD600 = 1 from WT, Hnm1 mutant yeasts or transformed yeasts was used for total protein extraction. BCA assay was used for total protein quantification. These antibodies were used: VDAC (Cell Signaling, CS4866), CoxIV (Invitrogen, MA5-15686), OPA1 (BD biosciences, 612602), MRPS35 (Protein biotech, 16457), CHKA (Cell Signaling, CS13422S), HMOX1 (Proteintech, 10701-1-AP), NDUFS1 (Proteintech, 12444-1-AP). Western blot analysis was conducted as previously described.2 (link)
6
Immunoblotting Analysis of Mitochondrial Proteins
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Proteins from lysates were prepared and resolved by 12% SDS-PAGE at 100 V, transferred for 1.5 h at 250 mA onto Nitrocellulose membranes, and analysed by immunoblotting as described previously56 (link). The information for primary antibodies is as follows: anti-ferritin (cat# 69090), SdhA (cat# 137040), and SdhB (cat# 178423) from Abcam (Cambridge, MA, USA), anti-Xod (cat# 55156-1-AP), citrate synthase (cat# 16131-1-AP), aconitase 2 (Aco2) (cat# 11134-1-AP), Ndufs1 (cat# 12444-1-AP), Ndufs3 (cat# 15066-1-AP), Uqcrc1 (cat# 21705-1-AP), and Uqcrfs1 (cat# 1843-1-AP) from Proteintech Group Inc. (Chicago, IN, USA), anti-Actin (cat# BM0627) from Boster (Wuhan, China), anti-Tubulin (cat# T0198) from Sigma-Aldrich (St. Louis, MO, USA), anti-TfR1 antibody (cat# 136800) from Zymed (San Francisco, CA, USA), anti-IscU, Fech, Irp1, and Irp2, anti-Fxn (self-made)31 (link), anti-CytC (cat# 1896-1) from Epitmics (Burlingame, CA, USA), anti-Vdac (cat# 4661S) from Cell Signaling Technology Inc (Shanghai, China).
7
Western Blot Analysis of Cellular Proteins
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The harvested cells were lysed in 1% NP-40, 125 mM Tris, pH 6.8, containing a protease inhibitor cocktail (Roche, Basel, Switzerland). For western blotting, 25–50 µg total protein was loaded into 10–12% SDS-PAGE per lane and analyzed by immunoblotting as described previously [46 (link)]. The primary antibodies included: TfR1 antibody (cat#136,800) from Zymed (San Francisco, CA), SDHB (cat#ab178423), cleaved-caspase3 (cat#ab179517) from Abcam (Cambridge, MA), ACO2 (cat#11134-1-AP), NDUFS1 (cat#12444-1-AP), UQCRFS1 (cat#1843-1-AP), NRF2 (cat#16396-1-AP), HO-1 (cat#66743-1-Ig) from Proteintech Group Inc. (Chicago, IN), anti-FTL, FTH, IRP1, and IRP2 (polyclonal, self-made, raised from rabbits) [47 (link)].
8
Immunoblotting of Mitochondrial Proteins
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Equal amounts of protein per well were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were incubated with primary antibodies raised against the following proteins: β-Actin (1:3000, Cat#ab20272, Abcam), CD63 (1:1000, Cat#ab68418, Abcam), TSG101 (1:1000, Cat#ab125011, Abcam), CD9 (1:1000, Cat#ab236630, Abcam), CD81 (1:1000, Cat#ab79559, Abcam), Calnexin (1:1000, Cat#ab22595, Abcam), PARP1 (1:1000, Cat#ab32064, Abcam), Caspase3 (1:1000, Cat#ab32351, Abcam), Cleaved-Caspase-9 (1:1000, Cat#ab2324, Abcam), Bcl-2 (1:1000, Cat#ab182858, Abcam), Bcl-xl (1:1000, Cat#ab32370, Abcam), Bax (1:1000, Cat#ab32503, Abcam), Bad (1:1000, Cat#ab32445, Abcam), Cytochrome C (1:1000, Cat#ab133504, Abcam), AIF (1:1000, Cat#ab32516, Abcam), COX-IV (1:1000, Cat#ab33985, Abcam), Drp1 (1:1000, Cat#ab184247, Abcam), Phospho-Drp1 (1:1000, Cat#3455, CST), Fis1 (1:1000, Cat#ab96764, Abcam), Opa1 (1:1000, Cat#ab157457, Abcam), Ndufs1 (1:1000, Cat#12444-1-AP, Proteintech), Sdha (1:1000, Cat#14865-1-AP, Proteintech), Mfn1 (1:1000, Cat#14739, CST), Uqcrc1 (1:1000, Cat#21705-1-AP, Proteintech), ATP5A (Cat#14676-1-AP, Proteintech), Mfn2 (1:1000, Cat#11925, CST) and Ki67 (1:1000, Cat# ab16667, Abcam).
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