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Hsp60

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The HSP60 is a lab equipment product manufactured by Merck Group. It is a heat shock protein that functions as a molecular chaperone, assisting in the folding and unfolding of other proteins within the cell.

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Lab products found in correlation

Gapdh, by Santa Cruz Biotechnology (2 mentions) Actin, by Merck Group (2 mentions) β actin, by Merck Group (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) Ubiquitin, by Santa Cruz Biotechnology (1 mentions) Ab14748, by Merck Group (1 mentions) Atp5a, by Abcam (1 mentions) Cox5b, by Santa Cruz Biotechnology (1 mentions) Cox17, by Proteintech (1 mentions) Ndufs1, by Santa Cruz Biotechnology (1 mentions) Timm9, by Abcam (1 mentions) Tomm20, by Santa Cruz Biotechnology (1 mentions) Yme1l, by Proteintech (1 mentions) Orotate, by Merck Group (1 mentions) Uridine, by Merck Group (1 mentions) Teriflunomide, by Merck Group (1 mentions) Suloctidil, by Merck Group (1 mentions) Terfenadine, by Merck Group (1 mentions) Ivermectin, by Merck Group (1 mentions) Cytochrome c, by BD (1 mentions)

12 protocols using hsp60

1

Mitochondrial Protein Expression Analysis

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The antibodies against following proteins were used in the study: actin (Sigma, A1978, 1:500), ALR (Santa Cruz Biotechnology, Sc‐134869, 1:500), ATP5A (Abcam, ab14748, 1:500), COA7 (Sigma, HPA029926, 1:500), COX4 (Abcam, ab14744, 1:500, and Cell Signaling Technology, 4850, 1:2,000), COX5B (Santa Cruz Biotechnology, Sc‐374417, 1:500), COX6A (Rabbit Serum, 3282.7, 1:1,000), COX6B (Abcam, ab110266, 1:500), COX17 (Proteintech, 11464‐1‐AP, 1:100), GAPDH (Santa Cruz Biotechnology, Sc‐47724, 1:1,000), HSP60 (Sigma, H4149, 1:500), MIA40 (Rabbit Serum, WA136‐5, 1:500), MIC60 (Novus Biologicals, NB100‐1919, 1:1,000), NDUFS1 (Santa Cruz Biotechnology, Sc‐50132 1:1,000), SDHB (Santa Cruz Biotechnology, Sc‐25851, 1:500), TIMM9 (Abcam, ab57089, 1:200), TIMM22 (Proteintech, 14927‐1‐AP, 1:500), TOMM20 (Santa Cruz Biotechnology, Sc‐11415, 1:500), SDHA (Santa Cruz Biotechnology, Sc‐166947, 1:1,000), YME1L (Proteintech, 11510‐1‐AP, 1:100), ubiquitin (Santa Cruz Biotechnology, Sc‐8017, 1:500), UQCR1 (Sigma, HPA002815, 1:500).
Mohanraj K., Wasilewski M., Benincá C., Cysewski D., Poznanski J., Sakowska P., Bugajska Z., Deckers M., Dennerlein S., Fernandez‐Vizarra E., Rehling P., Dadlez M., Zeviani M, & Chacinska A. (2019). Inhibition of proteasome rescues a pathogenic variant of respiratory chain assembly factor COA7. EMBO Molecular Medicine, 11(5), e9561.
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2

Mitochondrial Dynamics Regulation Assay

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Apogossypol and Leflunomide from ApexBio (Boston, MA, USA), A-1331852, A-1210477, ABT-199, Z-VAD.FMK and CCCP from Selleck (Houston, TX, USA), Teriflunomide, norhydroguaiaretic acid (NDGA), Ivermectin, Terfenadine, Suloctidil, orotate and uridine from Sigma Aldrich (St Louis, MO, USA), MitoTracker Deep Red FM from Thermo Fisher (Loughborough, UK) were used. Antibodies against BAP31, RTN4, BiP, PDI, CHOP, DHODH and tubulin from Abcam (Cambridge, UK), CLIMP-63 and BAX (6A7) from Enzo Life Sciences (Exeter, UK), TIM22 and KNT-1 from Sigma, HSP60, Cytochrome c, BAX, OPA1 and DRP-1 from BD Biosciences (San Jose, CA, USA), phospho-DRP-1 (S616), phospho-DRP-1 (S637), MFN1 and MFN2 from Cell Signaling Technologies (Danvers, MA, USA), BAK (AB-1) from Calbiochem (Watford, UK), MFF, MID49 and MID51 from ProteinTech (Manchester, UK) and GAPDH from Santa Cruz Biotechnologies (Santa Cruz, CA, USA) were used. For RNA interference, cells were transfected with 10 nM of siRNAs against DHODH (SI00363384 and SI00363391) purchased from Qiagen Ltd. (Manchester, UK), using Interferin (Polyplus Transfection Inc, NY), according to the manufacturer’s protocol and processed 72 h after transfection.
Yedida G., Milani M., Cohen G.M, & Varadarajan S. (2019). Apogossypol-mediated reorganisation of the endoplasmic reticulum antagonises mitochondrial fission and apoptosis. Cell Death & Disease, 10(7), 521.
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3

Comprehensive Antibody Panel for Western Blotting

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Primary antibodies: Actin (Millipore, MAB1501, 1:10 000), DAXX (Sigma-Aldrich, D7810-2ML, 1:1000), DAXX (Cell Signaling Technology, #4533, 1:1000), ATRX (Abcam, ab97508, 1:1000), GAPDH (Cell Signaling Technology, #5174, 1:7000), RNase H1 (Abcam, ab229078, 1:1000), BRCA1 (Bethyl, A300-000A, 1:1000), GFP (Roche, 11814, 1:1000), Vinculin (Santa Cruz, sc-73614, 1:2000), HSP60 (Sigma-Aldrich, H3524, 1:2000), KAP1 (Abcam, ab10483,1:1000), SETDB1 (Proteintech,11231–1-AP,1:1500), Histone H3.3 (Abcam, ab176840, 1:1000), Histone H4 (Cell Signaling Technology, #13919, 1:1000). Secondary antibodies: HRP rabbit (Jackson Laboratory, Cat. No. 111-035-003, 1:50 000), HRP mouse (Amersham/Sigma, Cat. No. GENA931-1ML, 1:10 000).
Pinto L.M., Pailas A., Bondarchenko M., Sharma A.B., Neumann K., Rizzo A.J., Jeanty C., Nicot N., Racca C., Graham M.K., Naughton C., Liu Y., Chen C.L., Meakin P.J., Gilbert N., Britton S., Meeker A.K., Heaphy C.M., Larminat F, & Van Dyck E. (2023). DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks. Nucleic Acids Research, 52(3), 1136-1155.
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4

Quantitative PCR Analysis of Gene Expression

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qPCR was carried out to assess transcript expression of genes of interest. Amplifilour UniprimerTM Universal system (Intergen company, New York, USA) was utilised for qPCR [29 (link)]. In brief, each reaction was made up with 5 μL precision FAST2x qPCR Master Mix (PrimerDesign, Southampton, UK), 0.3 μL forward primers (EPLIN: AAGCAAAAATGAAAACGAAG; GAPDH: AAGGTCATCCATGACAACTT; Her1: GACCTCCATGCCTTTGAGAA; Her2: CCTCCTCGCCCTCTTG; Her3: CCCCACACCAAGTATCAGTA; Her4: CTGCTGAGTTTTCAAGGATG; HSP60: TGTAGACCTTTTAGCCGATG), 0.3 μL reverse primer with z sequence whose concentration was 1/10 of forward primer (EPLIN: ACTGAACCTGACCGTACAGACACCCACCTTAGCAATAG; GAPDH: ACTGAACCTGACCGTACAGCCATCCACAGTCTTCTG; Her1: ACTGAACCTGACCGTACAGCACAAATTTTTGTTTCCTGA; Her2: ACTGAACCTGACCGTACACATGTCCAGGTGGGTCT; Her3: ACTGAACCTGACCGTACAACACAGGATGTTTGATCCAC; Her4: ACTGAACCTGACCGTACAAACTTGCTGTCATTTGGACT; HSP60: ACTGAACCTGACCGTACAACAGTCACACCATCTTTTGT) (Sigma-Aldrich Co, Poole, Dorset, UK), 0.3 μL uniprimer and 4.1 μL cDNA samples. In addition to the test cDNA samples, a set of known transcript copy number samples (ranging from 101 to 108) was also run as standard relative copy number of cDNA samples were calculated based on this standard and normalised to the housekeeping control, GAPDH.
Zeng J., Sanders A.J., Ye L., Hargest R., Ruge F, & Jiang W.G. (2022). EPLIN, a Putative Tumour Suppressor in Colorectal Cancer, Implications in Drug Resistance. International Journal of Molecular Sciences, 23(23), 15232.
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5

Western Blot Protein Expression Analysis

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The detection of protein expressions in cells and tissues were performed by Western blotting as described previously. Cells and tissues were lysed in the buffer containing 150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris-HCl (pH 8.0). The protein samples were separated by SDS-PAGE and transferred onto the Immobilon P membranes (Millipore Technology, Billerica, MA, USA). After blocking with for 5% skin milk solution for 2 hours, the membranes were incubated overnight at 4°C with primary antibodies for p27Kip1 (1:1000; Santa Cruz Biotechnology, Inc., SC, CA, USA), AGEs (1:2000; ab23722, Abcam, Cambridge, MA, USA), HSP60 (1:3000), RAGE (1:2000), and lamin A (1:3000) (Sigma-Aldrich). Follow, the secondary antibody were incubated for 1 hour and the membranes were detected by using enhanced chemiluminescence (ThermoFisher, Con, CO, USA) on LAS-4000mini performing system (Fuji Film, Tokyo, Japan). The blotting bands were quantified by densitometric analysis using Multi Gauge v3.2 software (Fuji Film, Tokyo, Japan).
Guan S.S., Sheu M.L., Yang R.S., Chan D.C., Wu C.T., Yang T.H., Chiang C.K, & Liu S.H. (2016). The pathological role of advanced glycation end products-downregulated heat shock protein 60 in islet β-cell hypertrophy and dysfunction. Oncotarget, 7(17), 23072-23087.
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6

Antibody Characterization in Cell Biology

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The following antibodies were used collagen VII (rabbit anti–human [Abcam]; mouse anti–human [Sigma-Aldrich]), ERGIC-53 (mouse anti–human; Santa Cruz Biotechnology, Inc., and Enzo Life Sciences), Sec31A (mouse anti–human; BD), TANGO1 (rabbit anti–human; Sigma-Aldrich; rabbit anti-human in-house), HSP47 and calreticulin (goat anti–human; Enzo Life Sciences), HA (mouse; BioLegend), SAR1 (mouse anti–human; Abcam), β-tubulin (mouse anti-human; SIGMA-Aldrich), β-actin (mouse anti-human; SIGMA-Aldrich), NBAS (rabbit anti-human SIGMA-Aldrich), RINT1 (rabbit anti-human; SIGMA-Aldrich and goat anti-human (Santa Cruz Biotechnology), ZW10 (rabbit anti-human; Abcam), Sec23 (rabbit anti-human/mouse/rat; Abcam), cTAGE5 (rabbit anti-human Atlas antibodies, mouse anti-human Santa Cruz Biotechnology), TGN46 (sheep polyclonal, Bio-Rad), HA (mouse monoclonal, BioLegend; rat monoclonal BioLegend), FLAG (mouse monoclonal, rabbit, SIGMA-Aldrich; goat, Novus) HSP60 (mouse anti-human SIGMA-Aldrich), c-myc (mouse monoclonal, rabbit, SIGMA-Aldrich). Mounting media used in confocal and STED microscopy were either Vectashield (Vector Laboratories) or ProLong (Thermo Fisher Scientific, Waltham, Massachusetts).
Raote I., Ortega-Bellido M., Santos A.J., Foresti O., Zhang C., Garcia-Parajo M.F., Campelo F, & Malhotra V. (2018). TANGO1 builds a machine for collagen export by recruiting and spatially organizing COPII, tethers and membranes. eLife, 7, e32723.
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7

Exosome-mediated myeloid cell differentiation

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Mouse bone marrow cells were obtained and co-cultured with IL-6 (40 ng/mL, PeproTech) plus GM-CSF (40 ng/mL, PeproTech), LDEVs or exosomes for 4 days in a 24-well plate.8 (link) Then the phenotype and percentage of these cells were detected in flow cytometric applications. The following Abs were used for flow cytometry analysis of mouse cells: CD11b-FITC, Ly6G-PE, Ly6C-APC, Gr-1-APC (Biolegend). In some cases, 10μg LDEVs were pre-treated with HSP27, HSP60, HSP70, HSP90, HMGB1 antibodies (10 μg, Sigma Aldrich) separately for 24h in 4°C and then washed three times with PBS for follow-up study.
Gu Y., Liu Q., He Q., Wu Q., Li L., Xu D., Zheng L., Xie L., Cheng S., Shen H., Zhou Y., Yang J., Jin H, & Zhang X. (2024). LC3-dependent extracellular vesicles promote M-MDSC accumulation and immunosuppression in colorectal cancer. iScience, 27(5), 109272.
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8

Immunoblotting of Mouse Heart Proteins

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Mouse hearts were homogenized (1:10 w/v) in RIPA buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 12 mM deoxycholic acid, 0.5% Nonidet P-40, and protease and phosphatase inhibitors). Fibroblasts (1 × 106) were lysed on ice with RIPA buffer, including DTT and protease inhibitors. Fourty μg proteins were subjected to SDS PAGE on 4–12% denaturing gel and probed with the following antibodies: NRF2 (1:1000, Thermo Fisher Scientific, USA), GPX4 (1:1000, Thermo Fisher Scientific, USA), Frataxin (1:500, Santa Cruz Biotechnology, USA), Hsp60 (1:10000) and vinculin (1:10000, Sigma) as loading controls. Immunoreactive bands were detected using the Lite Ablot Extend Long Lasting Chemiluminescent substrate (Euroclone, Milan, Italy). Signals derived from appropriate HRP-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) were captured by Chemi DocTM XRS 2015 (Bio-Rad Laboratories, Hercules, CA, USA) and densitometric analysis was performed using Image Lab software (Version 5.2.1, Bio-Rad Laboratories).
La Rosa P., Petrillo S., Turchi R., Berardinelli F., Schirinzi T., Vasco G., Lettieri-Barbato D., Fiorenza M.T., Bertini E.S., Aquilano K, & Piemonte F. (2020). The Nrf2 induction prevents ferroptosis in Friedreich's Ataxia. Redox Biology, 38, 101791.
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9

Platelet Activation and Mitochondrial Response to Amyloid-β

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Platelets were activated with collagen-related peptide (= CRP, Richard Farndale, University of Cambridge, United Kingdom), synthetic Aβ40 (1-40; Bachem, Switzerland, cat no 4014442.1000) sequence single-letter code (DAEFRHDSGYEVHHQKLVFFAEDVGSN-KGAIIGLMVGGVV), Aβ16 (Aβ1-16, Bachem, Switzerland) ADP (Sigma-Aldrich). Apyrase (grade II, from potato) and prostacyclin from Calbiochem were used for isolation. Antimycin A (Streptomyces sp., A8674-25MG, Sigma-Aldrich, St. Louis, USA) was solved in 95% EtOH. Vitamin C (L(+)-Ascorbic acid) is from VWR Chemicals. Antibodies: Hsp60 (SAB 4501464; Sigma Aldrich, dilution 1:1000), OPA1 (sc-393296, Santa Cruz, dilution 1:500), PINK1 (D8G Rabbit mAb 6946; Cell Signalling, dilution 1:500), TIM23 (BD 611222; BD Biosciences, dilution 1:500), TOM20 (sc-11415; Santa Cruz, dilution 1:500), Amyloid-β (6E10, SIG-39320; Covance, dilution 1:2000). The antibodies β-actin (cat no 4967) and horseradish peroxidase (HRP)-linked secondary antibodies (cat no 7074 and cat no 7076) were from Cell Signaling Technology.
Donner L., Feige T., Freiburg C., Toska L.M., Reichert A.S., Chatterjee M, & Elvers M. (2021). Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet–Mediated Amyloid Aggregation in Alzheimer’s Disease. International Journal of Molecular Sciences, 22(17), 9633.
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10

Antibody Validation for Western Blotting

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The following primary antibodies and dilutions were used in this study: ALAS2 (Abcam, ab184964), 1:1000; CLPX (Abcam, ab168338), 1:1000; CLPP (Abcam, ab12482), 1:1000; FECH (EMD Millipor Corp, ABS2124), 1:500; PPOX (Abnova, H00005498-M01), 1:500; HSP60 (Abcam ab59457) 1:750; HSP60 (Sigma, A5316), 1:750; b-actin (Santa Cruz, sc47778), 1:1000; SDHB (Abcam ab14714), 1:1000; MFRN1 (Proteintech, 26469-1-AP), 1:500. Fluorescent secondary antibodies were obtained from Li-COR and probed at a dilution of 1:10,000. Secondary antibodies for chemiluminescence were from Sigma and used at a dilution of 1:10,000.
Rondelli C.M., Perfetto M., Danoff A., Bergonia H.A., Gillis S., Nicolas G., Puy H., West R., Phillips J.D., & Yien Y.Y. (2020). CLPX regulates erythroid heme synthesis by control of mitochondrial heme synthesis enzymes and iron utilization.
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