Hsp60

Manufactured by Cell Signaling Technology

Sourced in United States, Germany

HSP60 is a protein that functions as a molecular chaperone, facilitating the proper folding and assembly of other cellular proteins. It is involved in the transportation and refolding of unfolded or misfolded proteins within the mitochondria.

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Lab products found in correlation

64 protocols using hsp60

Western Blotting and Immunocytochemistry of Cellular Markers

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The following commercially available antibodies were used for Western blotting: TNAP (R&D Systems, AF2910), OXPHOS (Abcam, ab110413), UCP1 (Abcam, ab10983), VDAC (Abcam, ab34726), COX1 (Abcam, ab14705), HSP60 (Cell Signaling, 12165), and VCL (Sigma-Aldrich, V9264). The following commercially available antibodies were used for immunocytochemistry: TNAP (R&D Systems, MAB29092), HSP60 (Cell Signaling, 12165), OXPHOS (Abcam, ab110413), Gt-anti-Ms Alexa Fluor 568 (Invitrogen, A-11004), and Gt-anti-Rb Alexa Fluor 647 (Invitrogen, A-21245).

Sun Y., Rahbani J.F., Jedrychowski M.P., Riley C.L., Vidoni S., Bogoslavski D., Hu B., Dumesic P.A., Zeng X., Wang A.B., Knudsen N.H., Kim C.R., Marasciullo A., Millán J.L., Chouchani E.T., Kazak L, & Spiegelman B.M. (2021). Mitochondrial TNAP Controls Thermogenesis by Hydrolysis of Phosphocreatine. Nature, 593(7860), 580-585.

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Western Blot Analysis of Esophageal Tissue

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Frozen rat esophagus tissue was homogenized in Tissue Protein Extraction Reagent (ThermoFisher, Waltham, MA, USA) using TissueLyser II (Qiagen) at 30 Hz for 5 min. Extracted protein was then quantified using the DC Protein Assay (Bio-Rad). Western blot analysis was performed as previously described [123 (link)]. Images were captured using the Bio-Rad ChemiDoc Imaging System and quantified by means of chemiluminescent immunodetection using Bio-Rad Image Lab Software version 6.1.0 with expression levels normalized to the loading control GAPDH. Immunoblotting was performed using commercially available antibodies from Abcam (Cambridge, MA, USA) and Cell Signaling Technology (Danvers, MA, USA): ABST (ab203205, 1:500), ATF-4 (CST #11815, 1:1000), CD44 (ab189524, 1:500), GAPDH (CST #2118, 1:25,000), HSP60 (CST #12165, 1:5000), IRE1α (CST #3294, 1:1000), and phospho-AMPK (CST #2535, 1:1000). Patient EAC samples with matched normal and BE tissues were collected at the University Hospital at the University of Michigan. Informed consent was obtained from patients prior to sample collection. Protein extraction and quantification were similarly performed as described above.

Zhang Y., Weh K.M., Tripp B.A., Clarke J.L., Howard C.L., Sunilkumar S., Howell A.B, & Kresty L.A. (2023). Cranberry Proanthocyanidins Mitigate Reflux-Induced Transporter Dysregulation in an Esophageal Adenocarcinoma Model. Pharmaceuticals, 16(12), 1697.

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Mitochondrial Protein Analysis by Western Blot

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Fibroblasts were lysed in RIPA buffer containing 1 × complete protease inhibitor (Roche). Western blot analysis was performed with antibodies against Miro1 (WH0055288M1; Sigma–Aldrich, Munich, Germany), LC3-I/II (2775; Cell Signaling), Hsp60 (4870; Cell Signaling), Tom20 (sc-17764; Santa Cruz Biotechnologies), Rab9 (sc-74482; Santa Cruz Biotechnologies), MnSOD (ab13533; Abcam), anti-V5 (R960-25; Novex, R96125; Sigma–Aldrich), and β-actin (MA1-744; Thermo Scientific). Mitochondrial fractionation was performed as described previously (12 (link)).

Grossmann D., Berenguer-Escuder C., Bellet M.E., Scheibner D., Bohler J., Massart F., Rapaport D., Skupin A., Fouquier d'Hérouël A., Sharma M., Ghelfi J., Raković A., Lichtner P., Antony P., Glaab E., May P., Dimmer K.S., Fitzgerald J.C., Grünewald A, & Krüger R. (2019). Mutations in RHOT1 Disrupt Endoplasmic Reticulum–Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease. Antioxidants & Redox Signaling, 31(16), 1213-1234.

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Comprehensive Mitochondrial Protein Analysis

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Several primary antibodies were used: OPA1 (612607; BD Biosciences), PDH (ab110333; Abcam), GAPDH (MA5-15738; Thermo), HSP60 (12165; Cell Signaling), TOM20 (11802-1-AP; Proteintech), MPC1 (14462; Cell Signaling Technology), MPC2 (46141; Cell Signaling Technology), CS (14309; Cell Signaling Technology), ACO2 (6571; Cell Signaling Technology), DRP1 (611113; BD Biosciences), MFF (Gandre-Babbe and van der Bliek, 2008 (link)), IDH2 (56439; Cell Signaling Technology), Fumarase (4567; Cell Signaling Technology), SDHA (11998; Cell Signaling Technology), SDHB (ab14714; Abcam), SDHC (14575-1-AP; Proteintech), SDHD (6847; ProSci), ubiquitin (3933S; Cell Signaling Technology), and p62 (GP62-C; Progen). Secondary antibodies were purchased from Invitrogen: Alexa 488 anti-Rabbit IgG (A21206), Alexa 488 anti-Mouse IgG (A21202), Alexa 568 anti-mouse IgG (A10037), and Alexa 647 anti-mouse IgG (A31571).

Yamada T., Murata D., Kleiner D.E., Anders R., Rosenberg A.Z., Kaplan J., Hamilton J.P., Aghajan M., Levi M., Wang N.Y., Dawson T.M., Yanagawa T., Powers A.F., Iijima M, & Sesaki H. (2022). Prevention and regression of megamitochondria and steatosis by blocking mitochondrial fusion in the liver. iScience, 25(4), 103996.

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Mitochondrial Protein Immunoblotting

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Aliquots of total liver or heart mitochondrial protein were electrophoresed on Criterion SDS polyacrylamide gels (BioRad, Hercules, CA) and transferred to nitrocellulose membranes. Primary antibodies used for immunoblotting were: anti-TFPα (Abcam, 1:1000), TFPβ (Santa Cruz, 1:250), anti-SCHAD (Abcam, 1:2000), anti-acetyllysine (Cell Signaling, 1:1000), anti-succinyllysine (PTM Biolabs, 1:1000) and anti-heat shock protein-60 (Hsp60, Cell Signaling, 1:2000) as a mitochondrial marker for loading control. After incubation with HRP-conjugated secondary antibodies (1:5000) blots were visualized with chemiluminescence. The blots were scanned and subjected to densitometric analysis using ImageJ software, normalized to either Hsp60 or to Ponceau S membrane staining.

Zhang Y, & Goetzman E. (2021). The enzyme activity of mitochondrial trifunctional protein is not altered by lysine acetylation or lysine succinylation. PLoS ONE, 16(10), e0256619.

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Mitochondrial Protein Analysis by Western Blot

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Mitochondrial proteins were separated by SDS-PAGE (12% resolving gel) and transferred to PVDF membranes. After blocking in 5% milk, the membranes were incubated with antibodies against LDHB (1:2000, Abcam), GAPDH with HRP-conjugated (1:4000, Cell Signaling), ALDH2 (1:4000, Invitrogen), or HSP60 (1:2000, Cell Signaling) overnight at 4 °C. After incubation with HRP-linked secondary antibodies (anti-mouse, 1:2500, Cell Signaling; anti-rabbit, 1:2500, Cell Signaling; and anti-goat, 1:2500; Invitrogen), the membranes were developed using Pierce™ ECL Western Blotting Substrate (ThermoFisher). Immunoreactive proteins were imaged using a BioRad™ ChemiDoc™ MP Imager.

Fulghum K.L., Rood B.R., Shang V.O., McNally L.A., Riggs D.W., Zheng Y.T, & Hill B.G. (2019). Mitochondria-associated lactate dehydrogenase is not a biologically significant contributor to bioenergetic function in murine striated muscle. Redox Biology, 24, 101177.

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Immunoblotting Analysis of Cellular Proteins

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Cellular protein extracts were prepared using RIPA lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, and 0.1% SDS). Protein lysates were subjected to immunoblotting using antibodies against SOD1, poly ADP ribose polymerase (PARP, BD Bioscience Pharmingen, San Diego, CA), caspase-3, caspase-8, p53, p21, MCL-1, Bcl_xL, c-Myc, cytochrome-c (Santa Cruz Biotechnology), caspase-9, p-eIF2α (Abcam, Cambridge, MA), cyclin-B1, CDC25C, CDC2, HSP60, CLPP, COX IV, PERK, BIP, Calnexin, GFP (Cell Signaling, Beverly, MA), polyubiquitin (Enzo Life Sciences, Inc., Farmingdale, NY), GAPDH, or β-actin (Sigma-Aldrich).

Du T., Song Y., Ray A., Chauhan D, & Anderson K.C. (2020). Proteomic analysis identifies mechanism(s) of overcoming bortezomib resistance via targeting ubiquitin receptor Rpn13. Leukemia, 35(2), 550-561.

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Inflammasome Protein Detection Protocol

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Protein levels of inflammasome signaling proteins were determined in the cytosolic and mitochondrial fractions as described in [7 (link)]. Briefly, protein lysates were resolved in 10–20% Criterion TGX Stain-Free precasted gels (Bio-Rad), using antibodies (1:1000 dilution) to NLRC4 (Novus Biologicals, cat# NBP2–41124), caspase-1 (Novus Biologicals, cat# NB100–56565), caspase-11 (Novus Biologicals, cat# MAB8648), ASC (Santa Cruz, cat# sc-271054), IL-1β (Cell Signaling, cat# 12242S), IL-18 (Abcam, cat# ab71495), HSP60 (Cell Signaling, cat#12165) and β-actin (Sigma Aldric, cat# A5441). Quantification of band densities was done using the UN-SCAN-IT gel 6.3 Software (Silk Scientific Corporation). Chemilluminescence substrate (LumiGlo, Cell Signaling) was imaged with the ChemiDoc Touch Imaging System (BioRad).

Mejias N.H., Martinez C.C., Stephens M.E, & de Rivero Vaccari J.P. (2018). Contribution of the inflammasome to inflammaging. Journal of Inflammation (London, England), 15, 23.

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Immunoblotting Analysis of Liver Proteins

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Protein immunoblotting was performed as previously described [30 (link)]. Briefly, frozen, pulverized liver was homogenized in radioimmunoprecipitation buffer (RIPA; 150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris, pH 8.0) and centrifuged at 4 °C, 1,000× g to produce a post-nuclear supernatant. Protein concentration of the lysate was determined using BCA protein assay, and equal protein was loaded and resolved using Bolt Bis-Tris Plus gels prior to transfer to nitrocellulose membranes for incubation with antibodies as noted below. Immunoblots were developed with Licor Odyssey CLx using Licor IRDye goat secondary antibodies and analyzed using Licor Image Studio Software (Version 5.2). Primary antibodies were as follows: PARKIN (Santa Cruz sc-32282), OXPHOS antibody cocktail (Abcam ab110413), HSP60 (Cell Signaling 12165D), VDAC (Cell Signaling 4661S), SDHA (Cell Signaling 11998S), PHB1 (Cell Signaling 2426S), phospho-AKT^SER473 (Cell Signaling 4060), AKT (Cell Signaling 2920), and GAPDH (Santa Cruz sc-25778 and sc-365062).

Edmunds L.R., Xie B., Mills A.M., Huckestein B.R., Undamatla R., Murali A., Pangburn M.M., Martin J., Sipula I., Kaufman B.A., Scott I, & Jurczak M.J. (2020). Liver-specific Prkn knockout mice are more susceptible to diet-induced hepatic steatosis and insulin resistance. Molecular Metabolism, 41, 101051.

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Immunoblotting Experimental Protocol

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Immunoblotting experiments were conducted as previously described (15 (link)). Antibodies used for immunoblotting were specific for the following proteins: LC3b, PERK, IRE1α, P-AMPK, AMPK (total), P-ULK (Ser⁷⁵⁷), P-ULK (Ser³¹⁷), ULK (total), COXIV, cytochrome c, calnexin, HSP60, mitofusin-1, and Bcl-2 (Cell Signaling Technology); BAP31, ATF6, Tom40, Tom22, prohibitin, and VDAC1 (Santa Cruz Biotechnology); Parkin and FACL-4 (Abcam); and NDUFS4, NDUFB11, α-tubulin, β-actin, and flag (Sigma-Aldrich). Antibodies were diluted to 1:1000, except for NDUFS4 (1:500) or anti–β-actin (1:10,000). Secondary antibodies were purchased from Promega (anti-rabbit and anti-mouse at 1:5000).

, & Namba T. (2019). BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites. Science Advances, 5(6), eaaw1386.

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