Mitochondrial fractionation of NCI‐H1048 or NCI‐H69 monolayer and spheroid cells were extracted by using Mitochondria/Cytosol Fractionation Kit (ab65320, Abcam, Cambridge, MA, USA) according to the manufacturer's protocol. Briefly, SCLC monolayer cells and spheroid cells were collected and washed with PBS. Cells were then resuspended in cytosol extraction buffer and incubated on ice for 10 minutes, followed by homogenization for non‐mitochondrial fractionation extraction. After centrifugation, mitochondrial components were collected and resuspended in mitochondrial extraction buffer. Both mitochondrial fractionations and non‐mitochondrial fractionations were used for immunoprecipitation analyses. Mitochondrial COX IV (1:1000; ab202554, Abcam) and GAPDH (1:10000; HRP‐60004, Proteintech, Wuhan, China) were used as loading control [37 (link)].
Ab202554
Manufactured by Abcam
Sourced in China, United Kingdom, United States
Ab202554 is a rabbit polyclonal antibody that recognizes Methylene tetrahydrofolate reductase. This antibody is suitable for use in Western Blot applications.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mitochondria cytosol fractionation kit, by Abcam (1 mentions)
Hrp 60004, by Proteintech (1 mentions)
Ab182032, by Abcam (1 mentions)
Ab109025, by Abcam (1 mentions)
Ab182422, by Abcam (1 mentions)
Anti cd163 antibody, by Abcam (1 mentions)
Ab23707, by Abcam (1 mentions)
Ab218311, by Abcam (1 mentions)
Ab51243, by Abcam (1 mentions)
Enhanced chemiluminescence method, by Cytiva (1 mentions)
Ab77924, by Abcam (1 mentions)
Sc 126, by Santa Cruz Biotechnology (1 mentions)
L7543, by Merck Group (1 mentions)
Sc 48402, by Santa Cruz Biotechnology (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Ab190828, by Abcam (1 mentions)
Ab239511, by Abcam (1 mentions)
Ab157208, by Abcam (1 mentions)
11 protocols using ab202554
1
Mitochondrial Fractionation of SCLC Cells
2
Immunohistochemical Characterization of Inflammatory and Metabolic Markers
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Formalin-fixed and paraffin-embedded WT sliced samples were collected from AHMU. CD14 (Anti-CD14 antibody, Cat. ab182032, Abcam Inc., Massachusetts, USA) and CD163 (anti-CD163 antibody, Cat. ab182422, Abcam Inc., Massachusetts, USA) were chosen to reflect the infiltration of monocytes and macrophages [23] , [24] (link), [25] , [26] , [27] . The expression level of MMP9 was examined by anti-MMP9 antibody (bs-0397R, Bioss Inc, Beijing, China). COX1 (anti-COX1 antibody: ab109025, Abcam Inc., Massachusetts, USA) and COX4 (anti-COX4 antibody: ab202554, Abcam Inc., Massachusetts, USA) were chosen to reflect the oxidative phosphorylation level. Following the same procedures as have been reported previously [28] (link), [29] (link), positively stained cells were counted by ImageJ software (NIH, Bethesda, USA) [30] (link).
3
Autophagy and Senescence Markers Analysis
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At 0, 24 and 48 hours, NPCs were collected and lysed in lysis buffer (Beyotime) on ice using a western and IP cell lysis kit. Then, the protein extracts were collected by centrifugation at 15 000 g for 15 minutes at 4°C. Protein concentrations of cell lysates were tested using an enhanced BCA protein assay kit (Beyotime). After protein transfer, the membranes were blocked with nonfat milk and then incubated overnight at 4°C with rat antibodies against LC3B (L7543, 1:1000, Sigma), p62 (sc‐48402, 1:500, Santa Cruz, USA), p16 (ab51243, 1:1000, Abcam), p21 (ab218311, 1:1000, Abcam), p53 (sc‐126, 1:500, Santa Cruz), PARKIN (ab77924, 1:200, Abcam), PINK1 (ab23707, 1:500, Abcam), COX IV (ab202554, 1:1000, Abcam) and β‐actin (8H10D10, 1:1000, CST). After three washes, the membranes were incubated using peroxidase‐conjugated secondary antibodies for 1 hour at room temperature. Finally, the proteins were visualized using the enhanced chemiluminescence method following the manufacturer's instructions (Amersham Biosciences).
4
Protein Expression Analysis of Oxidative Stress
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The protein extracts prepared after sample collection were detected by WB. The oxidative stress–associated protein nuclear factor–erythroid 2-related factor-2 (Nrf-2; ab92946, Abcam), lipid peroxidation–associated protein malondialdehyde (MDA; ab27642, Abcam), mitochondrial injury–associated protein cytochrome c oxidase IV (COXIV; ab202554, Abcam), heat shock protein 60 (HSP60; ab190828, Abcam), system Xc−–associated protein solute carrier family 7, member 11 (SLC7A11; ab175186, Abcam), glutathione peroxidase-4 (GPX4; ET1706-45, HUABIO), ferroptosis-associated protein ferroportin-1 (FPN1; ab239511, Abcam), and divalent metal transporter 1 (DMT1; ab157208, Abcam) were used, with β-actin (AF7018, Affinity, England) as the control. Goat anti-rabbit IgG (Biosharp, China) was used as the secondary antibody. A western blotting ECL kit (ZETA-Life, San Francisco, CA, USA) was used. After the detection step, protein bands were observed and photographed using an automated chemiluminescence image analysis system (Tanon, Shanghai, China), and various parameters were quantified using ImageJ software (National Institutes of Health, DC, USA). The procedure was repeated three times before statistical analysis using SPSS 26.0.
5
Mitochondrial Protein Isolation and Analysis
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Protein extraction from cybrids for sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting was carried out as described previously (Ji et al., 2020 (link)). Briefly, cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, United States). The protein extracts were resolved in a 10% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% milk for 1 h and incubated with the following primary antibodies at 4°C overnight: anti-MT-CO3 (cat #ab110259; Abcam, Cambridge, United Kingdom) and VDAC (cat #4661; Cell Signaling Technology, Danvers, MA, United States); the latter served as a loading control. Blue native (BN)-PAGE was performed with mitochondrial proteins isolated from cybrids as described previously (Delmiro et al., 2013 (link)), using antibodies against MT-COXIV (ab202554; Abcam), SDHA (ab14715; Abcam), VDAC (cat#4661; Cell Signaling Technology), UQCRC2 (ab14745; Abcam), NDUFS2 (ab110249; Abcam), and ATPB (ab14730; Abcam). The in-gel activity (IGA) of CIV was performed as described previously (Jha et al., 2016 (link)). Briefly, after running the native PAGE, incubate the gel in 20 ml of complex IV substrate. Appearance of brown bands is indicative of CIV activity.
6
Western Blot Profiling of Cellular Signaling
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Western blotting was carried out using an SDS-PAGE Electrophoresis System. Adherent cells or adipose tissue extracts were prepared and transferred to PVDF membranes. The primary antibodies for this experiment were as follows: anti-GAPDH (AB2000, Abways, Shanghai, China), anti-Col XV (ab202554, Abcam, Shanghai, China), anti-CHOP (YM3668, Immuno Way, Suzhou, China), anti-GRP78 (ab21685, Abcam, Shanghai, China), anti-IRE1α (ab124945, Abcam, Shanghai, China), anti-IL-6 (ab100712, Abcam, Shanghai, China), anti-MCP1 (ab100712, Abcam, Shanghai, China), anti-IL-1β (ab100712, Abcam, Shanghai, China), anti-CD206 (ab125028, Abcam, Shanghai, China), anti-CD163 (YM6146, Immuno Way, Suzhou, China), anti-TNFα (11948P, Cell signaling, Massachusetts, USA), anti-pFAK (ab81298, Abcam, Shanghai, China), anti-FAK (ab40794, Abcam, Shanghai, China), anti-Integrin β1 (ab24693, Abcam, Shanghai, China), Horseradish peroxidase anti-rabbit (Sigma-Aldrich, Shanghai, China) or anti-goat (Sigma-Aldrich, Shanghai, China) was used as secondary antibody. See Antibody Information Sheet (Appendix A ) for antibody details.
7
Protein Expression Analysis in Cells
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The kidneys, cells, or EVs pellets were lysed in Radio-Immune Precipitation Assay (RIPA) lysis buffer containing phenylmethylsulphonyl fluoride (PMSF), and the supernatant suspension obtained after centrifugation was measured using a BCA protein assay kit (Thermo Fisher Scientific). Protein samples of the same quality (20 μg/lane) were added to a 10% or 12% sodium dodecyl sulphate–polyacrylamide gel (SDS-PAGE) and then transferred from the SDS-PAGE gels to the membranes. The membrane was blocked for 2 h at room temperature with a 1X casein solution, and then incubated with primary antibodies at 4 °C against the following proteins: α-SMA (ab7817, Abcam), vimentin (ab92547, Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 60004-1-Ig, Proteintech), actin (20536-1-AP, Proteintech), PGC1α (ab191838, Abcam), CPT1A (ab128568, Abcam), succinate dehydrogenase complex iron sulphur subunit B (SDHB, 10620-1-AP, Proteintech), cytochrome c oxidase subunit IV (COXIV, ab202554, Abcam), and ATPB (17247-1-AP, Proteintech). The membrane was then incubated with the corresponding secondary antibody for 1.5 h at room temperature and washed three times. The images were analysed using ImageJ software.
8
Western Blot Analysis of Mitochondrial Proteins
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The following antibodies were used for western blotting analysis: rabbit monoclonal anti-uncoupling protein 2 (UCP2) (#89326, Cell Signaling Technology, United States; 1:1,000 dilution), rabbit polyclonal anti-uncoupling protein 3 (UCP3) (#BA1722, Boster Biological Technology, United States; 1:500 dilution), rabbit polyclonal anti-cytochrome C (Cyt C) (#ab90529, Abcam, United Kingdom; 1:1,000 dilution), rabbit monoclonal anti-mitochondrial transcription factor A (mtTFA) (#ab252432, Abcam, United Kingdom; 1:1,000 dilution), rabbit monoclonal anti-Cyt C oxidase subunit IV (COX IV) (#ab202554, Abcam, United Kingdom; 1:2,000 dilution), rabbit monoclonal anti-caspase 3 (#ab184787, Abcam, United Kingdom; 1:5,000 dilution), rabbit polyclonal anti-cleaved-caspase 3 (#9661, Cell Signaling Technology, United States; 1:1,000 dilution). Secondary antibodies and other reagents not specified were obtained from Sigma-Aldrich (Darmstadt, Germany).
9
Protein Extraction and Western Blot Analysis
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Total protein from cells or brain tissues was isolated by RIPA buffer (Beyotime). In addition, mitochondria were isolated from the cytoplasm using the Cell Mitochondria Isolation Kit (Beyotime) and incubated with the mitochondrial lysis buffer supplemented with PMSF (Beyotime) to extract mitochondrial proteins. After quantification using BCA Protein Quantification Kit (Thermo Fisher Scientific), equal amounts of proteins were loaded onto an SDS-PAGE gel for separation. Subsequently, the protein signal was transferred onto the PVDF membrane and blocked by 5% non-fat milk powder. The membrane was incubated with primary antibodies against SENP1 (1/1000; AF0275, Affinity), NeuN (1/2000; ab177487, Abcam), GFAP (1/10000; ab7260, Abcam), LC3 (1/2000; ab192890, Abcam), p62 (1/10000, ab109012, Abcam), OPTN (1/1000; ab213556, Abcam), COX IV (1/2000; ab202554, Abcam) and GAPDH (1/10000; ab181602, Abcam) overnight at 4°C. After incubating with appropriate secondary antibody at 37°C for 1 h, the blot was visualized using ECL detection system (Thermo Fisher Scientific). Relative protein levels were assessed by ImageJ software.
10
Immunoblotting of Apoptosis Regulators
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Briefly, cells (0.4 × 106) were homogenized in ice-cold lysis buffer. After centrifugation at 5,000 g for 30 min, the protein content of the supernatant was quantified using a Bradford protein assay. Samples were diluted, boiled with sample loading dye, and 100 µg were used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4561033EDU, Bio-Rad, USA). After blotting, membranes were blocked in 5% skim milk (70,166, Sigma-Aldrich) in tris-buffered saline containing 0.1% Tween-20 (P1379, Sigma-Aldrich). Membranes were incubated with Bcl-2 (1:1,000; ab59348, RRID: AB_289591, Abcam, UK), Bcl-xL (1:1,000; ab32370, RRID: AB_2847960, Abcam, UK), cytochrome c oxidase subunit IV (COX IV) (1:1,000; ab202554, RRID: AB_2847960, Abcam, UK), Bid (1:1,000; ab32060, RRID: AB_365478, Abcam, UK), cytochrome C (1:1,000; #11,940, RRID: AB_2637071, Cell signaling technology, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000; ab8245, RRID: AB_1267174, Abcam, UK) or beta-tubulin (1:1,000; ab179513, RRID: AB_1566837, Abcam, UK), and subsequently with secondary goat anti-rabbit horseradish peroxidase-conjugated antibody. Reaction products were visualized using an enhanced chemiluminescence detection kit (32106, Thermo Scientific, USA) and quantified by densitometry.
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