Cycloheximide-chase assays
WebApr 10, 2024 · Cycloheximide Pulse-Chase Assay. For the protein half-life assay, the indicated cells were treated with CHX (50 µg mL −1) for the indicated time points, and subjected to immunoblotting. Immunoprecipitation and In Vivo Ubiquitination Assay. WebApr 18, 2016 · The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. …
Cycloheximide-chase assays
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WebDec 6, 2024 · Scale bar = 20 μm. (D) Representative Western blot of cycloheximide chase assay showing increased stability of p53 in HeLa cells infected with control (Null) adenovirus or adenovirus expressing constitutively active TFEB and TFE3. (E) Quantification of cycloheximide chase assay shown in D. Values represent mean p53 protein levels ± … WebMay 28, 2024 · Cycloheximide chase assay The protein degradation rate of GPX4 was tested in HL-60 cells after treatment with cyclophosphamide (10 μM) in the absence or presence of cycloheximide (10 μM) for 1–4 h. Whole cell lysates were analyzed for GPX4 and ACTB by Western blot analyses.
WebAug 26, 2024 · Cycloheximide Chase Assay SpCENP-A NT mutant overexpression strains were pre-inoculated and incubated in EMM-Leu media supplemented with 100 μM of thiamine overnight at 30 °C with shaking. The induction of exogenous protein expression in log-phase strains bearing CENP-A N -terminal truncation plasmid constructs was … WebFeb 5, 2024 · Current loss of signal (“down”) assays for identifying degraders often exhibit poor signal-to-noise ratios, narrow dynamic ranges, and false positives from compounds that nonspecifically suppress transcription or translation. Here, we describe a gain of signal (“up”) assay for degraders.
WebCycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. Among 11,747 quantified proteins, we ... WebHere we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model. Materials A. Cell lines CL1-5/Flag-wtSlug stable cells CL1-5/Flag-4SA stable cells B. Antibodies Flag M2 antibodies (Sigma-Aldrich, catalog number: F3165) β–actin antibodies (Sigma-Aldrich, catalog number: A5441)
WebCycloheximide is a translation elongation inhibitor that immobilizes ribosomes (Godchaux et al., 1967; McKeehan and Hardesty, 1969), thereby preserving a snapshot of their location at the time of cycloheximide addition. From: Methods in Enzymology, 2010 Related terms: Glucosamine Maltose Lactose Protein Biosynthesis Gluconic Acid Raffinose
WebNational Center for Biotechnology Information the carpenter in little chute wiWebApr 7, 2024 · The stability of AUF1 and its degradation by ubiquitin-proteasome pathway were examined by cycloheximide chase analysis and co-immunoprecipitation assay. The regulation of AUF1 on nuclear factor E2-related factor 2 (NRF2) and activation transcription factor 3 (ATF3) was explored by RNA immunoprecipitation (RIP), RNA pull-down, and … tattoos symbolizing strength for womenWebCycloheximide (CHX) (Sigma Aldrich) is stored as a 500 μg/mL stock solution, solved in dimethylsulfoxide (DMSO) at − 20°C in small aliquots. Proteasome inhibitor MG132 … the carpenter hotel austin txWebThis chapter describes in detail the two most frequently used methods, pulse-chase analysis and cycloheximide blocking, to determine a protein’s half-life in yeast and cultured mammalian cells. ... Immunoprecipitation , SDS-PAGE, Bradford Protein Assay, Protein Assay, Autoradiography, Electrophoresis, Western Blot, Gel Electrophoresis. Models ... tattoos tacoma waWebCycloheximide is known to inhibit protein synthesis of symbiotic Chlorella of the ciliate P. bursaria preferentially, but it only slightly inhibits host protein synthesis (Ayala and Weis, … the carpenter hypixelWebLuciferase assays were carried out in 96-well white solid bottom plates. d -Luciferin (50 μl of 1 mg/ml in PBS) was added into each well containing 50 μl of medium and incubated at room temperature with shaking at 500 … the carpenter hospice of northwest louisianaWebNov 18, 2024 · Cycloheximide, a translational inhibitor, is widely used in targeted studies to measure degradation kinetics for short-lived proteins. Here, we combined cycloheximide chase assays with advanced quantitative proteomics to map short-lived proteins under translational inhibition in four human cell lines. the carpenter homeboy sandman lyrics