Computational protocol: Isopeptide and ester bond ubiquitination both regulate degradation of the human dopamine receptor 4

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Protocol publication

[…] Confocal images of PLA-treated neurons were acquired with a Nikon A1RSi point scanning confocal inverted microscope using a 60× oil immersion objective. Excitation lasers of 405, 488, and 640 nm were used. Images from single confocal planes are shown. Image processing (cropping and adding of scale bars) was done in NIS Elements. The brightness and intensity of the images were adjusted in Adobe Photoshop CS6 to enhance the possibility to observe fine structures on printouts, and the final figure was made in Adobe Illustrator CS6.For quantitative PLA in HEK293T cells, 20× magnification Z-stack images (every 1.5–2 μm) were captured using an FSX100 Olympus microscope. Exposure times were held constant while imaging samples within each experimental replicate. A total of four fields of view were obtained per sample per experiment. [...] All image processing was done using ImageJ. Nuclei stained with DAPI were counted to obtain the total number of cells per image. Then the number of infected cells, identified as nuclei surrounded by a full ring of 1D4 immunofluorescence, were counted. The total number of PLA dots per field was determined by creating a maximum projection of the PLA channel, converting it to a 32-bit grayscale image, and recording the number of dots as the number of PLA signals.The average number of PLA signals per cell of a control-infected plate was used to estimate background signal for each experiment. For each field to be quantified, the average number of background PLA dots per cell (averaged over the four images obtained from the control infection) was multiplied by the total number of cells in that field. This number was then subtracted from the total number of PLA dots in that field to remove background. The remaining dots were divided by the number of infected cells to account for variable infection efficiencies, resulting in the average number of PLA dots per infected cell per field. For each of the five biological replicates there were four fields per sample, resulting in a total of 20 data points per sample. Using Prism, a one-way ANOVA followed by Tukey's multiple comparison test was used to compare the mean of each group with that of every other group. Significance was determined by a p value <0.05. […]

Pipeline specifications

Software tools Adobe Illustrator, ImageJ
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Chloroquine, Dopamine