Data from "Dual-specificity phosphatase 5 (DUSP5) controls the localized inhibition, propagation and transforming potential of ERK signaling"

Data collection method:

Details of experimental procedures can be found in the Kidger et al. 2017 publication. Detailed high content microscopy methodology can also be found in the following paper: Caunt, Kidger and Keyse, Methods Mol Biol. 2016;1447:197-215. doi: 10.1007/978-1-4939-3746-2_12. Data provided in the Microsoft Excel file include a combination of data derived from high content microscopy and automated analysis (GE Healthcare IN Cell 2000 microscope and IN Cell Developer software), immunoblot analysis from scanned images using a Licor Odyssey scanner, quantitative PCR techniques and a small number of manual counting assays for cellular senescence and focus formation.

Data processing and preparation activities:

For high content microscopy, single cell data is shown as raw arbitrary fluorescence unit (AFU) values. For population-averaged data, where immunostaining experiments were pooled from several independent experiments, each of which usually had several replicate samples per plate, average AFU values from entire wells were normalised as percentages of values from control wells in each plate and applied across all technical replicate wells in the 96-well plate, prior to data pooling between independent experiments. Other types of data derived from high content microscopy are represented as discrete values. For example, an AFU cutoff for label intensity above background values was used to assign whether a cell had entered S-phase within a given pulse window of fluorescent labelling. All analysis of nuclear and cytoplasmic intensity values was carried out using a custom routine described in Caunt, Kidger and Keyse, Methods Mol Biol. 2016;1447:197-215. doi: 10.1007/978-1-4939-3746-2_12. Where possible, we have included Western blot as original blot scans, as well as raw intensity values in the spreadsheet derived from the Licor scanner, and also values corrected for loading and normalised to control samples contained in each replicate blot (or normalised according to the mean signal intensity across the blot).

Technical details and requirements:

Please see Kidger et al 2017 for details of experimental procedures and equipment used.

Methodology link: