Nuclear extracts and whole cell extracts were prepared using NUM buffer as described in Reinke et al.18 (link) or Nuclear/Cytosol Fractionation Kit (BioVision). Immunoblotting was carried out as described previously31 (link). The antibodies used were anti-Sp1 (07-645, Millipore), anti-Actin (sc-8432) and anti-USF1 (sc-229, Santa Cruz Biotechnology), anti-HSF1 (#4356, Cell Signal Technology), and anti-HSP70 (SPA-812) and anti-HSC70/HSP70 (SPA-822, StressGen).
Anti hsf1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-HSF1 is a laboratory reagent that detects the heat shock transcription factor 1 (HSF1) protein. HSF1 is a key regulator of the cellular stress response and is involved in the transcriptional activation of heat shock genes. The Anti-HSF1 product can be used to identify and quantify the HSF1 protein in various biological samples through techniques such as Western blotting, immunoprecipitation, and immunohistochemistry.
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Lab products found in correlation
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Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Ly294002, by Merck Group (3 mentions)
Anti ulbp 2 5 6 phycoerythrin, by Cell Signaling Technology (3 mentions)
Anti ulbp 1 alexa fluor 488, by Cell Signaling Technology (3 mentions)
Anti ulbp 3 allophycocyanin, by Cell Signaling Technology (3 mentions)
Anti p53 primary antibodies, by Cell Signaling Technology (3 mentions)
Metformin hydrochloride, by Merck Group (3 mentions)
Anti ddr1, by Cell Signaling Technology (3 mentions)
Anti sp1, by Merck Group (2 mentions)
Anti iba1, by Fujifilm (2 mentions)
Cryostat, by Leica (2 mentions)
Anti gfap, by Merck Group (2 mentions)
Anti neun, by Merck Group (2 mentions)
Anti β amyloid clone 4g8, by Fortrea (2 mentions)
Anti histone h3, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
24 protocols using anti hsf1
1
Protein Extraction and Immunoblotting Protocol
2
Immunohistochemical Analysis of Liver Lesions
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Liver lesions were fixed in 4% paraformaldehyde overnight at 4°C, embedded in paraffin, and evaluated by two board-certified pathologists (S.R. and F.D.) in accordance with the criteria by Frith et al [37 (link)]. For immunohistochemistry, antigen retrieval was performed in 10mM sodium citrate buffer (pH 6.0) by placement in a microwave on high for 10 min, followed by a 20 min cool down at room temperature. After a blocking step with 5% goat serum and Avidin-Biotin Blocking Kit (Vector Laboratories, Burlingame, CA), the slides were incubated with primary antibodies overnight at 4°C. Specifically, the anti-c-Myc (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA; # sc-40) and anti-HSF1 (Cell Signaling Technology Inc., Danvers, CA, USA; # 4356) primary antibodies were selected since they have been extensively validated by the manufacturers for immunohistochemistry. Slides were then subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and subsequently the biotin conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. The immunoreactivity was visualized with the Vectastain Elite ABC kit (Vector Laboratories), using Vector NovaRED™ (Vector Laboratories) as the chromogen. Slides were counterstained with Mayer’s hematoxylin.
3
Histological Analysis of Mouse Brain
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Mice were heart-perfused by saline and 4% PFA, postfixed in 4% PFA overnight. Mouse brains were paraffin-embedded and 5 μm sections were deparaffinized by passing through 100% xylene and rehydrated through serial dilutions of ethanol (100, 95, and 75%). Hematoxylin and Eosin (H&E) staining was performed and images were captured under a light microscope. For OCT embedding, perfused brains were cryoprotected by 30% sucrose infiltration and then sectioned at 10 μm using a cryostat (Leica). Anti-HSF1 (1:100; cell signaling), anti-β-Amyloid (clone 4G8; 1:100; Covance), anti-NeuN (1:100; Millipore), anti-GFAP (1:1000; Sigma), anti-Iba1 (1:500; Wako) antibody was used and nuclei were stained with DAPI.
4
Immunoblotting Analysis of Cellular Stress Responses
5
Myocardial Protein Analysis via Western Blot
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Myocardium tissues were harvested for Western Blot following standard protocol. Nucleoprotein was extracted according to the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Institute of Biotechnology, Suzhou, China). 50 μg/lane protein or 20 μg/lane nucleoprotein Proteins were run on 10% SDS-PAGE, and then electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated with the primary antibodies anti-HSF1(#4356; 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Omi/HtrA2(#2176; 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH(#2118; 1:1000; Cell Signaling Technology, Danvers, MA, USA), or anti-Histone H3 (#4499; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After incubation with the corresponding secondary antibodies, the membrane was developed with a chemiluminescent substrate (Bio-Rad), and protein bands were measured using the ImageJ software.
6
Immunohistochemistry for Key Signaling Proteins
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Immunohistochemistry was performed as described previously.36 (link) Primary antibodies were used including anti-β-catenin (1:1000, BD,
610154), anti-HSF1 (1:200, Cell Signaling Technology, No. 12972), anti-LKB1
(1:100, Santa Cruz, sc-32245), and anti-PCNA (1:1000, Santa Cruz, sc-7907).
610154), anti-HSF1 (1:200, Cell Signaling Technology, No. 12972), anti-LKB1
(1:100, Santa Cruz, sc-32245), and anti-PCNA (1:1000, Santa Cruz, sc-7907).
7
Western Blot and ELISA Protocols
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For western blot detection, the crude proteins were extracted and the concentration of proteins was determined using a Micro BCA protein assay kit (Thermo Fisher, USA). The cell lysates (30 µg/Lane) were separated on 10-12% gels using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins in the gel were then transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% fat-free dry milk in TBST, membranes were probed with primary antibodies, including anti-HSF1 (diluted 1:1000), anti-CD69 (diluted 1:500), and anti-β-actin (diluted 1:2500). The anti-HSF1 (Clone: D3L8I, Catalog: 12972S) and anti-β-actin (Clone: 8H10D10, Catalog: 3700S) antibody were purchased from Cell Signaling Technology. The anti-CD69 antibody (Clone: D-3, Catalog: sc-373799) were purchased from Santa Cruz. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz, USA) and visualized by enhanced chemiluminescent reagents (Thermo Fisher, USA).
The levels of cytokines such as IL-10 and TGF-β1 in the supernatant of cultured cells were measured using ELISA kits (eBioscience, USA). For the detection of TGF-β1, 100 µl of supernatants were acidified with 20 µL of 1N HCl at room temperature for 10 min and then neutralized with 20 µl of 1N NaOH to activate latent TGF-β1 into its immunoreactive form.
The levels of cytokines such as IL-10 and TGF-β1 in the supernatant of cultured cells were measured using ELISA kits (eBioscience, USA). For the detection of TGF-β1
8
Western Blotting for Protein Detection
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Proteins were resolved by SDS/PAGE and immunodetected with the following primary antibodies: anti‐HSF1 (4356, Cell Signaling Technology, Danvers, MA, USA), anti‐HSF2 (H300) (sc‐13056, Santa Cruz Biotechnology, Dallas, TX, USA), anti‐Nrf2 (D1Z9C) (12721, Cell Signaling Technology). Anti‐TFIID (TBP, 58C9 sc‐421 Santa Cruz Biotechnology) and/or anti‐specificity protein 3 (Sp3) (sc‐644, Santa Cruz Biotechnology) were used as nuclear loading control, while anti‐actin (A 2066, Sigma‐Aldrich) was used to demonstrate equal protein loading in whole cell extracts. Briefly, gels were electroblotted onto a nitrocellulose membrane (0.2 mm pore size) (Bio‐Rad, Hercules, CA, USA). The blots were probed with the primary antibodies listed above and bands were detected by horseradish peroxidase (HRP)‐conjugated secondary antibody (Bio‐Rad). Peroxidase activity was detected with the enhanced chemiluminescence detection method (WesternBright ECL, Advasta, Menlo Park, CA, USA).
9
Immunoblot Analysis of Protein Targets
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Immunoblots were carried out as previously described (25 (link)). A total of 20 μg of protein extracts from each sample were denatured in 4× Laemmli sample buffer (Biorad, 161–0747) and loaded into a Sodium dodecyl sulphate-polyacrylamide gel for immunoblot analysis. Immunoblots were performed using anti-ACTB (Sigma, A2066), anti-HSF1 (Cell Signaling, 4356), anti-MYC Tag (Cell Signaling, 2278), anti-HSPA1A (Cell Signaling, 4872) and anti-FOXO3 (Cell Signaling, 2497). Immunoblots were developed with the ECL-plus chemiluminescence reagent (GE Healthcare, RPN2232) according to the manufacturer’s instructions.
10
Quantifying Lipid Metabolism Pathways
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Anti-HSF1 (#12972), β-actin (#4970), anti-AMPKα (#5831), anti-phospho-AMPKα (50081), anti-rabbit anti-mouse HRP-linked secondary antibody IgG (#7074 and #7076) were purchased from Cell Signaling Technology. Anti-LRP1B (DF9609) was purchased from Affinity Bioscience. For lipid content measurement, a lipophilic fluorescent dye (DiD) (# V22887, ThermoFisher) and Oil Red O Stain kit (#YB0843, ScienCell) were used. Human FAS ELISA Kit (MS1108), ACC ELISA Kit (MS2411) and CPT-1A ELISA Kit (CSB-E17408h) were respectively from Shanghai Suobio Technology and Ximei Biotech.
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