Computational protocol: The human 18S rRNA base methyltransferases DIMT1L and WBSCR22-TRMT112 but not rRNA modification are required for ribosome biogenesis

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Protocol publication

[…] Immunofluorescence experiments were carried out on cells grown in 96-well plates. Cells were fixed in PBS/formaldehyde (4%) for 15 min at room temperature and rinsed three times for 5 min in PBS. The cells were then permeabilized by incubation in PBS supplemented with Triton X-100 (0.3%) and BSA (5%) for 60 min at room temperature. Primary antibodies targeting DIMT1L (ab69434; Abcam), WBSCR22 (HPA052185; Sigma-Aldrich), or TRMT112 (H00051504-M09; Novus Biologicals) were added at 1:500 in PBS supplemented with Triton X-100 (0.3%) and BSA (1%), and incubation was carried out for 16 h at 4°C. The cells were washed three times for 5 min in PBS before incubation for 1 h at room temperature with the appropriate secondary antibody (Alexa Fluor 488 or 594; Invitrogen) diluted at 1:1000 in PBS supplemented with Triton X-100 (0.3%) and BSA (1%). After washes in PBS, the cells were stained for 10 min at room temperature with 4′,6-diamidino-2-phenylindole (DAPI; 5 mg/ml, diluted 1:20,000; Sigma-Aldrich) and finally washed and stored in PBS before imaging. For lysosome or Golgi localization, cells were treated just before fixation, for 1 h at 37°C in the incubator, with Lyso- or Golgi-Tracker (LysoTracker Green DND-26 or CellLight Golgi-GFP BacMam 2.0; Life Technologies) diluted 1:20,000. The cells were washed once in PBS before being processed for immunofluorescence. Cells were observed with a Zeiss Axio Observer Z1 microscope driven by MetaMorph (MDS Analytical Technologies). High-resolution images were captured in the confocal mode with a Yokogawa spinning-disk head and the HQ2 camera with a laser illuminator from Roper (405-nm, 100-mW Vortran; 491-nm, 50-mW Cobolt Calypso; and 561-nm, 50-mW Cobolt Jive). To assess the specificity of the observed subcellular distribution of the relevant label, cells were incubated with secondary antibody alone and examined after the same exposure time. Images were analyzed with ImageJ (National Institutes of Health, Bethesda, MD). […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis
Organisms Homo sapiens, Saccharomyces cerevisiae
Chemicals Guanosine