Hsp90α

Manufactured by Cell Signaling Technology

Sourced in United States

Hsp90α is a protein that plays a crucial role in the proper folding and stabilization of various client proteins. It is a member of the heat shock protein 90 (Hsp90) family and is involved in a variety of cellular processes. The core function of Hsp90α is to act as a molecular chaperone, helping to ensure the correct three-dimensional structure and proper function of its client proteins.

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Anti-HSP90α (4 citations)

7 protocols using hsp90α

Evaluating Protein Ubiquitination and Expression

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Western blot and immunoprecipitation‐western blot analyses were carried out according to standard procedures using Immobilon‐P filters (Millipore) and an Amersham ECL western blotting detection reagent (GE Healthcare). Ubquitination assay was carried out as previously described.²¹ Antibodies against ROR1 (cat. no. 4102; Cell Signaling Technology), HSP90α (cat. no. ab2928; Abcam), HSP90β (cat. no. ab53497, Abcam), GRP94 (60012‐1‐Ig; Proteintech), p21 (cat. no. 2947; Cell Signaling Technology), LC3B (cat. no. 2775; Cell Signaling Technology), hemagglutinin (HA) (cat. no. M180‐3; MBL), Azami‐Green (hAG) (cat. no. PM011M; MBL), and cavin‐1 (cat. no. ab135655, Abcam; cat. no. A301‐271 A, Bethyl Laboratories) were used for western blot analysis. β‐Actin (cat. no. A5441; Sigma) was used as a loading control. For immunoprecipitation, anti‐goat IgG and immunoprecipitation‐specific anti‐ROR1 were used. Signal intensities of ROR1 protein bands were quantified using ImageJ (https://imagej.nih.gov/ij/) and normalized to β‐actin signal intensities.

Khaledian B., Taguchi A., Shin‐ya K., Kondo‐Ida L., Kagaya N., Suzuki M., Kajino T., Yamaguchi T., Shimada Y, & Takahashi T. (2021). Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma. Cancer Science, 112(3), 1225-1234.

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Immunoblot Analysis of Protein Expression

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For immunoblots, equal total protein of cell lysates (2.5–20 μg) were detected with antibodies to mouse p53 (FL393), human p53 (PAb1801; Santa Cruz Biotechnology), HSF1, p-Ser326 HSF1, AKT, pAKT, Erk, pErk, Hsp70, Hsp90, Hsp90α, EGFR, EGRF-Tyr845P (all Cell Signaling, Danvers, MA, USA), ErbB2, actin, GAPDH, GTS, GFP, HSc70 and HDAC1 (all Neomarkers, Fremont, CA, USA).^{35 (link), 36 (link), 37 (link)} SDS-PAAG gels (6%) were used to detect slower migrating HS-activated HSF1 used in experiments presented in Figures 2 and 3. SDS PAAG (10%) gels were used in all other experiments. Co-immunoprecipitations were performed with 1 μg of antibody overnight. Beads were washed with SNNTE and RIPA buffers 3 × each, followed by blotting.

Li D., Yallowitz A., Ozog L, & Marchenko N. (2014). A gain-of-function mutant p53–HSF1 feed forward circuit governs adaptation of cancer cells to proteotoxic stress. Cell Death & Disease, 5(4), e1194-.

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Immunoprecipitation of Protein Complexes

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Protein extraction from both yeast and mammalian cells was carried out using methods previously described (Mollapour et al., 2010 (link)). For immunoprecipitation, mammalian cell lysates were incubated with anti-FLAG or anti-HA antibody conjugated agarose beads (Sigma) for 2 h at 4°C. Immunopellets were washed 4 times with fresh lysis buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and eluted in 5x Laemmli buffer. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins were detected by immunoblotting with antibodies recognizing FLAG, 6x-His (ThermoFisher Scientific), Hsp90-835-16F1, GAPDH, p23 (ENZO Life Sciences), Tsc1, FLCN, GR, Myc, V5, GAPDH, Hsp90α, FNIP2, c-Src (Cell Signalling), phospho-tyrosine, v-Src (Millipore), FNIP1, FNIP2 (NCI), FNIP1 (antibodies-online.com), Aha1 (StressMarq Biosciences), HA (Roche). Secondary antibodies raised against mouse, rabbit, and rat (Cell Signaling) and goat (Santa Cruz Biotechnology) were used (See Key resources table).

Backe S.J., Sager R.A., Regan B.R., Sit J., Major L.A., Bratslavsky G., Woodford M.R., Bourboulia D, & Mollapour M. (2022). A specialized Hsp90 co-chaperone network regulates steroid hormone receptor response to ligand. Cell reports, 40(2), 111039.

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Immunoprecipitation of Protein Complexes

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Comprehensive Western Blot Methodology

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Western blotting was performed as described previously [24 (link)]. The primary antibodies were as followed: β-actin (66,009–1–1 g, PROTEINTECH), Bclaf1 (26,809–1-AP, Santa Cruz Biotechnology), VPS35 (sc-374,372, Santa Cruz Biotechnology), HSF1 (sc-9144, Santa Cruz Biotechnology), Hsp70 (sc-69,705, Santa Cruz Biotechnology), Hsp90α (8165 s, Cell Signaling Technology), TSG101 (sc-7964, Santa Cruz Biotechnology), CD63 (25,682–1-AP, PROTEINTECH). The secondary antibodies (Donkey anti-mouse/rabbit IRDye 680/800) were purchased from Li-COR Biosciences (Lincoln, Nebraska, USA). Blots were scanned by LI-COR Odyssey infrared imaging system and quantified with Image J software v1.8.0.

Tan W., Zhang J., Liu L., Liang M., Li J., Deng Z., Zheng Z., Deng Y., Liu C., Li Y., Xie G., Zhang J., Zou F, & Chen X. (2022). Hsp90 Inhibitor STA9090 induced VPS35 related extracellular vesicle release and metastasis in hepatocellular carcinoma. Translational Oncology, 26, 101502.

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Inhibition Assay for Topoisomerase and Hsp90

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A total of 720 compounds were purchased from the National Compound Resource Center, including 80 kinase inhibitors and 640 FDA−approved drug libraries. The positive control drugs etoposide (VP−16, Selleck, s1225), 17−AAG (Selleck, s1141), and quinacrine 2HCl (Selleck, s4255) were purchased as standards. All compounds were >95% pure by HPLC analysis. Geldanamycin−FITC (Abcam, ab141589), Topo IIα relaxation assay kit (Inspiralis, HTR202, UK), pBR322 DNA (Takara, 3050), topoisomerase I (Takara, 2240 A), and HSP90α (Abcam, ab80369) were purchased for the inhibitory assay. Antibodies phospho−ATM (Ser1981, no. 5883), phospho−Chk2 (Thr68, no. 2197), phospho−γH₂AX (Ser139, no. 9718), GAPDH (no. 5174), HSP90α (no. 8165), HSP70 (no. 4873), AKT (no. 4691), anti−rabbit IgG−HRP, and SignalFire™ elite ECL reagent (no. 12757) were obtained from Cell Signaling Technology. Annexin V−FITC and propidium iodide were obtained from KeyGEN BioTECH; 4′,6−diamidino−2−phenylindole dihydrochloride (DAPI, Thermo Fisher Scientific, no. D1306) and anti−rabbit Alexa 488−conjugated antibody (Abcam, ab150157) were purchase for the immunofluorescence assay.

Pan X., Mao T.Y., Mai Y.W., Liang C.C., Huang W.H., Rao Y., Huang Z.S, & Huang S.L. (2022). Discovery of Quinacrine as a Potent Topo II and Hsp90 Dual-Target Inhibitor, Repurposing for Cancer Therapy. Molecules, 27(17), 5561.

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Cell Lysis and Protein Analysis

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Lysis buffer containing 20 mM PIPES (pH 7.0), 100 mM NaCl, 2 mM Na₃VO₄, 20 mM Na₂MoO₄, 1 mM MgCl₂, 30 mM NaF, 1% Triton X-100 and protease inhibitors (Pierce) was used to lyse the cells and grind tissue. After centrifugation (14000 g) at 4 °C for 15 min (cells) or 30 min (tissue), the supernatant was collected and boiled with 5 × SDS-PAGE sample buffer. The pellets were resuspended in the same volume of 2 × SDS-PAGE sample buffer, sonicated on ice for 10s, centrifuged, and the supernatant collected as the insoluble protein fraction. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Primary antibodies: CDC37 (sc-13129), CDC25C (sc-327), Wee1 (sc-325), HSP70 (sc-69705) were from Santa Cruz Biotechnology, β-actin was from Tianjin Sungene Biotech Co, Cyclin B1 (#4138), CDK1 (#9112), p-CDK1 (Y15) (#9111), γ-H2AX (#9718), p21 (#2947), HSP90α (#8165), p-HSP90α (T5/T7) (#3488) were from Cell Signaling Technology, and p53 (A5761) was from Abclonal. IRDye secondary antibodies were from LI-COR Biosciences. Blots were scanned by Li-COR Odyssey infrared imaging system and analyzed by Image J 1.49 (NIH).

Huang Z., Zhou X., He Y., Ke X., Wen Y., Zou F, & Chen X. (2016). Hyperthermia enhances 17-DMAG efficacy in hepatocellular carcinoma cells with aggravated DNA damage and impaired G2/M transition. Scientific Reports, 6, 38072.

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