Abstract
Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.
Materials and Reagents
Equipment
Software
Procedure
Determine the time course of cycloheximide chase beforehand. There can be great variation among proteins. For an unfamiliar protein, it is recommended to start with a 4-hour interval and chase till 24 h. Cycloheximide may cause cytotoxicity to certain cells if cells are exposed to it over 20-24 h. It has been known that Slug is a labile protein with a T1/2 of about 40 min (Wang et al., 2009). CL1-5 cells stably overexpressing Flag-tagged wtSlug and non-phosphorylatable Slug-4SA were prepared using lentiviral transduction (Kao et al., 2014). Cells were grown in complete medium with 400 μg/ml G418. To compare the turnover of wtSlug and Slug-4SA, these stable cells were subjected to the cycloheximide chase assay.
Quantification
Quantification was achieved by the MetaMorph software in this case. An analysis guide should be referred to for a first-time user. Basically, regions of measurement are selected and threshold is defined. Draw region of interest on top of the western blot bands and quantitate its total grey signal (this can be done in Image J and any other image analysis software). The values of total thresholded areas are measured; a ratio of Flag-Slug to its β-actin is then calculated. The final Slug protein turnover rate at each time point is the percentage of Slug/β-actin at t = 0 of each experimental group. A plot can be created afterwards.
Representative data
Western blots and quantification images have been published in Oncogene (Kao et al., 2014). For details, please refer to Figure 2f at Kao et al. (2014).
Recipes
Acknowledgments
This work was supported by grants from the National Science Council, Taiwan (NSC99-2628-B-006-031-MY3 NSC101-2325-B-006-018, NSC100-2321-B-002-071 and NSC101-2321-B002-068), National Taiwan University, Taiwan (10R71601-2), and National Institute of Health, USA (R01-GM-094231, to AIN). SP Wang is supported by a Human Frontier Science Program long-term fellowship. This protocol was adapted from previous work published in Oncogene (Kao et al., 2014).
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.