Walsh et. al. - Cell Host and Microbe 2022

Published: 30 August 2022| Version 1 | DOI: 10.17632/hkvx8gx2ft.1
Stephen Walsh,


Raw western blot and representative confocal microscopy images used in the paper "The bacterial effector GarD shields Chlamydia trachomatis inclusions from RNF213-mediated ubiquitylation and destruction" by Walsh et al., 2022.


Steps to reproduce

For Western Blotting: 1. Open files in ImageJ. Files are hyperstacks of 4 individual images, and are saved directly from an Azure 500 Western Blot imaging machine (Azure Biosystems). (NOTE: the blot for CTL0390/GarD in figure S2B has two files per protein, one with chemiluminescent channel only and a separate PDF marking location of the ladder bands) 2. Click "Image"--> "Stack" --> "Stack to Images" to obtain four individual images. 3. The first two individual files (appended with "-0001" and "-0002") are blank and can be ignored (NOTE: the blot for RNF213 knockout pools in Figure S6C uses a different ladder (Precision Plus Protein Dual Color Standards #1610374) than all other blots. Images labeled "-0001" and "-0002" contain the red and blue bands for this ladder. Use these channels instead of the image labeled "-0003" in the steps below) 4. The third image (appended with "-0003") represents a photo taken of the protein ladder (Precision Plus Protein™ All Blue Prestained Protein Standards #1610373). 5. Invert ladder image by clicking "Edit"-->"Invert". 6. The last image in the file (appended with "-0004") corresponds to the chemiluminescent channel with the target protein 7. Invert target protein image by clicking "Edit"-->"Invert". 8. Minimally adjust brightness to view protein bands, or to check for the presence of fainter bands (e.g. total absence in KOs) 9. Merge target protein image with ladder image to gauge the size of the target protein by clicking "Image"-->"Color"-->"Merge Channels". Select desired colors for each image and keep the "Keep source images" option clicked 10. Check size of target protein according to ladder and compare to the predicted size of protein (e.g. UniProt or manufacturer's product page for antibody, see KRT in STAR methods) 11. Compare images with figure in paper *see Figure annotations for order of samples / treatment conditions For Microscopy: 1. Open confocal image files (.lsm) in ImageJ or Fiji; files represent unaltered images taken from a Zeiss 880 Airyscan Fast Inverted Confocal microscope and ZEN software 2. The individual channels should appear as a hyperstack; Channel contents are labeled within the file name 3. Press "Image"-->"Adjust"-->"Brightness and Contrast" to minimally adjust brightness of individual channels for visibility; pseudo-color individual fluorescent channels as desired 4. Using the rectangle tool, select an area of the field of view based on the inclusion(s) presented in the figure and press "Image"-->"Crop" 5. Add scale bar for size comparison ("Analyze"-->"Tools"-->"Scale Bar") 6. Press "Images"-->"Stacks"-->"Stack to Images" to obtain individual channels; convert colored images to grayscale using "Image"-->"Lookup Tables"-->"Grays" 7. Create a separate "Merge" image using the "Image"-->"Colors"-->"Merge Channels" tool 8. Compare with presented figure(s) in paper


Duke University


Microbiology, Cellular Aspects of Innate Immunity, Confocal Microscopy, Ubiquitins, Western Blot, Immunocytochemistry, Ubiquitylation, Chlamydia