direct interaction with ubiquitin E3 ligases9,32. To address whether XAF1 enhances the protein stability of IRF-1, a cycloheximide (CHX) chase experiment was performed using tetracycline-inducible XAF1 system (HCT116/Tet-XAF1). XAF1 induction by tetracycline addition led to an increase in the ...
5B). In addition, a cycloheximide chase assay revealed that the stability of DAXX protein was dramatically increased by CUL3 depletion (Fig. 5C), and an in vivo ubiquitination assay revealed that CUL3 and SPOP overexpression significantly increased the poly-ubiquitination of DAXX (Fig. 5D). If...
Cycloheximide chase assay was carried out as described (15, 19). Cells were incubated with 100 μg/ml cycloheximide in culture medium at 37 °C. At the times indicated in the figures, cells were harvested and lysed as described before (15, 19). Quantitative RT-PCR analysis was carried out...
The half-life of the PGAM5-L protein in the absence or presence of Keap1 was determined in a cycloheximide pulse-chase experiment. Transfected HeLa cells were treated with cycloheximide to block protein synthesis, and the level of the PGAM5-L protein was determined at time points after the ...
Cycloheximide chase assay showed that without DINO binding to p53, the p53 protein stability and p21 protein levels induced by DNA damage were decreased even with the preservation of p53 phosphorylation at Ser9. Furthermore, DINO overexpression can stimulate DNA damage signaling and stabilize p53. A...
Cycloheximide-chase assay Cells were treated with 10 µg/mL of cycloheximide (Sigma-Aldrich) and harvested at the indicated times. Samples collected at each time point were then analyzed by western blot. Mass spectrometry to determine MeCP2 PTMs All protein samples were digested overnight at ...
Cycloheximide DSB: DNA double-strand break ER: Estrogen receptor HR: Homologous recombination IR: Ionizing radiation KDM4B: Histone lysine demethylase KO: Knockout NHEJ: Nonhomologous end-joining RIPA: Radioimmunoprecipitation assay RSK: Ribosomal S6 kinase SERM: Selective estrogen receptor...
MG132, Cycloheximide (CHX), EGF protein, 10,058-F4, and gefitinib were obtained from MedChemExpress (MCE, NJ, USA). Puromycin were from Sigma-Aldrich (MO, USA). All information regarding antibodies used in this study is provided in Supplementary Table3. ...
We next measured VPS35 degradation rates by a cycloheximide (CHX) chase assay. Neuro2a cells received vehicle or compound 2a (10 µM) for 24 h, then CHX was added and VPS35 levels were measured by WB. As reported34, we observed that vehicle-treated cells exhibited a drop of VPS...
To measure if the enhanced E6AP activity due to P6 stimulation would accelerate the degradation of the substrate proteins in the cell, a cycloheximide (CHX) chase assay was done to follow the stability of UbxD8 and HHR23A in the presence of P6. HEK293 cells were pretreated with various conc...