Glutathione S-transferase (GST)-fused proteins (PPARγ domain mutants) immobilized with glutathione-agarose were incubated with TRIM25-expressing cell lysates for 2 h at 4 °C. Protein complexes were pulled down by centrifugation and washed four times with binding buffer. Precipitates were detected by immunoblotting using anti-GST or TRIM25 antibodies. For analyzing interactions between endogenous PPARγ and TRIM25, 3T3-L1 adipocytes were lysed with binding buffer. Cell lysates were incubated with anti-PPARγ or TRIM25 antibodies and analyzed by western blotting. HEK-293 cells expressing PPARγ, TRIM25, or their mutants were lysed in binding buffer, and total cell lysates were incubated with an anti-hemagglutinin (HA) antibody at 4 °C. Immunoprecipitants or total cell lysates were analyzed with specific antibodies as indicated. The antibodies used in this study included α-TRIM25, α-PPARγ, α-GST, α-Ub, α-aP2, and α-adipsin antibodies, which were purchased from Santa Cruz Biotechnology (Dallas, TX), while α-HA, α-actin, α-HSP90, and α-adiponectin were purchased from Cell Signaling Technology (Danvers, MA).
α hsp90
Manufactured by Cell Signaling Technology
α-HSP90 is a laboratory tool used to detect the expression of the heat shock protein 90 (HSP90) in biological samples. HSP90 is a molecular chaperone that plays a crucial role in the folding and stability of various client proteins. The α-HSP90 product allows researchers to analyze the levels and distribution of HSP90 in their experiments.
Automatically generated - may contain errors
Lab products found in correlation
Protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
α adipoq, by GeneTex (2 mentions)
Immobilon p transfer membrane, by Merck Group (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
α actin, by Cell Signaling Technology (1 mentions)
α pparγ, by Santa Cruz Biotechnology (1 mentions)
α gapdh, by Abcam (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Iscript, by Bio-Rad (1 mentions)
Faststart sybr green master, by Roche (1 mentions)
α flag, by Merck Group (1 mentions)
α gfp, by Santa Cruz Biotechnology (1 mentions)
α β tubulin, by Merck Group (1 mentions)
Pfa2 creb, by Agilent Technologies (1 mentions)
α ucp1, by Abcam (1 mentions)
α caspase 8, by Cell Signaling Technology (1 mentions)
αcleaved caspase 8, by Cell Signaling Technology (1 mentions)
5 protocols using α hsp90
1
PPARγ-TRIM25 Interaction Characterization
2
Cytoplasmic and Nuclear Protein Extraction
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Cells grown in 10-cm culture dishes were scraped in 1 ml PBS after media removal and spun at 500 g for 5 min at 4°C. Cells were lysed in 500 µl buffer A (150 ml NaCl; 50 mM Tris, pH 7.4; 0.01% Saponin; 1x Protease Inhibitor Cocktail [Thermo Fisher Scientific, Waltham, MA]), and incubated on ice for 10 min. Cytoplasm and nuclei were separated with a 10 min spin at 3000 g at 4°C, and both fractions were washed in 500 µl buffer A before an additional 10 min spin at 3000 g at 4°C. Nuclei were resuspended in 500 µl buffer A and sonicated 3x for 5 s. RNA was Trizol (Life Technologies) extracted from 100 µl lysate and reverse transcribed using iScript (Bio-Rad, Hercules, CA). qPCR was performed with FastStart SYBR Green Master (Roche, Indianapolis, IN), requiring at least one primer in each mRNA primer pair to be specific for an exon junction. qPCR results were normalized as indicated. Western blot analysis was performed using 15 µl cell lysate per lane, and the following antibodies: α-GAPDH (1:25:000; Abcam [Cambridge, MA] ab8245), Hu subject antiserum (1:1000 dilution), α-HSP90 (1:1000; Cell Signaling Technology [Danvers, MA] 4877S), α-H3 (1: 2000; Abcam ab1791), α-RNA PolII (1:1000; Millipore [Billerica, MA] 05–623), and α-RO60 (1:50; gift from S. Wolin). Immunoreactive bands were analyzed with the Odyssey Infrared Imaging System (LI-COR, Lincoln, NE) and normalized as indicated.
3
Molecular Cloning and Characterization of CREB
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Gal4-CREB (pFA2-CREB; Agilent) encodes the 147 amino acids Gal4 DNA-binding domain fused to CREB amino acids 1–280. KCREB was a gift from Dr Richard Goodman. GFP-CREB (249-341) and GFP-CREB (282–341) were subcloned into pQCXIN for GFP-Q2-bZIP and GFP-bZIP, respectively. 6×-His-CREB 1–197 was generated by digesting full-length CREB:pET-28a with KpnI followed by Klenow end polishing and religation. The GST-CRTC2CBD plasmid was a kind gift of Dr Marc Montiminy and has been described (12 (link)). FLAG-CREB expression plasmids in pFLAG-CMV and pcDNA3.1-based vectors have been described (20 (link),24 (link),25 (link)). Purified CREB protein was a kind gift from Dr Jennifer Nyborg (Colorado State University). Protein was extracted from cultured cells in high salt lysis buffer (25 mM HEPES (pH 7.4), 300 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100) supplemented with protease inhibitors and phosphatase inhibitors for 10 min on ice and proteins were separated on a SDS-PAGE. Antibodies used in this study include: α-pCREB-Ser-108/Ser-111/Ser-114 (24 (link)), α-pCREB-Ser-121 (20 (link)), α-pCREB-Ser-133 (Millipore 06–519 and Cell Signaling 87G3), α-FLAG (Sigma F7425), α-FUS (Santa Cruz 4H11), α-MCM3 (Bethyl laboratory A300-192A), α-Gal4 (Santa Cruz RK5C1), α-GFP (Santa Cruz B-2), α-β-tubulin (Millipore AA2) and α-HSP90 (Cell Signaling C45G5).
4
Multiparametric Immunoblotting of Cellular Metabolic Regulators
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Tissue and whole cell lysates were extracted using Protein Extraction Reagent (Thermo Fisher) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Immunoblotting was performed with lysates run on 4–12% Bis-Tris NuPage gels (Life Technologies) and transferred onto Immobilon-P Transfer Membranes (Millipore) followed by antibody incubation. Immunoreactive bands were visualized by chemiluminescence. The following antibodies were used for immunoblotting: α-ASPA (Abcam #154503), α-ADIPOQ (GeneTex #GTX112777), α-PPARG (Cell Signaling #2443), α-UCP1 (Abcam #ab10983), α-phospho-CAD S1859 (Cell Signaling #12662), α-CAD (Cell Signaling #11933), α-phospho-P70S6 Kinase T389 (Cell Signaling #9205), α-P70S6 Kinase (Cell Signaling #2708), α-DHODH (Proteintech #14877-I-AP), α-HSP90 (Cell Signaling #4877).
5
Immunoblotting Protein Expression Analysis
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Tissue and whole cell lysates were prepared in Protein Extraction Reagent (Thermo Fisher) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher). Immunoblotting was performed with lysates run on 4–12% Bis-Tris NuPage gels (Life Technologies) and transferred onto Immobilon-P Transfer Membranes (Millipore) followed by antibody incubation. Immunoreactive bands were visualized by chemiluminescence. The following antibodies were used for immunoblotting: α-HSP90 (Cell Signaling #4877), α-UBE2I (Cell Signaling #4786), α-ADIPOQ (Genetex #GTX112777), α-PPARγ (Cell Signaling #2443), α-Caspase-8 (Cell Signaling #4790), and α-Cleaved Caspase-8 (Cell Signaling #8592).
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