As a ribosome inhibitor, CHX can restrict the translation elongation of eukaryotic protein synthesis. Once protein synthesis is inhibited by CHX, the level of intracellular proteins decreases by degradation through the proteasome or lysosome system. Thus, the CHX chase assay is widely recognized and ...
the level of remaining Drp1 at different time points was quantified as the percentage of initial Drp1 level (0 hour of CHX treatment). C and D, the half-life of Drp1 in the presence of GFP-Parkin or GFP alone was analyzed by [35S] methionine pulse-chase assay, and the protein levels ...
S]methioninepulse-chaseassayfor thehalf-lifeofDrp1.A,293TcellsweretransfectedwithGFP-ParkinandGFP-Parkin(ΔR2) for24hours.CellswerethentreatedwithCHX(100μg/ml)fortheindicatedhours,and Westernblottingwasperformed.B,thelevelofremainingDrp1atdifferenttimepointswas ...
Cycloheximide (CHX) chase assay and [35S] methionine pulse-chase assay for the half-life of Drp1. A, 293T cells were transfected with GFP-Parkin and GFP-Parkin (ΔR2) for 24 hours. Cells were then treated with CHX (100 μg/ml) for the indicated hours, and Western blotting was ...
Harvest all cells at the same time point “0h CHX” with a standard trypsinization protocol and proceed as described inSection 4.2.3. View chapter Book series 2019,Methods in Enzymology Chapter Ubiquitin-dependent Protein Degradation 3.2Cycloheximide chase assay ...
(C) Flow cytometry spectra show similar results to luminescence assay. Shoulder peaks may indicate caspase-3 activity at earlier time points. (D) (Left) After treatment of cells with TNF-α/CHX, caspase-3 activation was evident as early as 30 min. This is indicated by the change in ...