Distinct host-dependent mimicry and autonomous translation termination strategies in marseilleviruses
Description
Western blotting analysis and immunofluorescence analysis of Acanthamoeba castellanii cells expressing foreign proteins.
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Antibodies Rabbit anti-FLAG primary monoclonal antibody (MAb), mouse anti-Myc primary MAb, horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary PAb and HRP-conjugated goat anti-rabbit secondary PAb were purchased from Epizyme. Rabbit anti-eRF3/GSPT1 primary MAb was purchased from Abcam (Cambridge, UK). Mouse anti-eRF1 primary MAb was purchased from Thermo Fisher Scientific. FITC-conjugated goat anti-mouse secondary PAb, FITC-conjugated goat anti-rabbit secondary PAb, CY3-conjugated goat anti-rabbit secondary PAb, CY3-conjugated goat anti-mouse secondary PAb, and DAPI were purchased from Servicebio (Wuhan, China). Western blot assay A. castellanii cells expressing foreign protein were grown in 6-well plates, which were divided into two groups, i.e., control groups (GFP or RFP) and viral protein groups (FTME476 or FTMF422). Foreign proteins were extracted from cell lysates in the ice-cold radioimmunoprecipitation buffer (EpiZyme; Shanghai, China) and quantified using the BCA Protein Assay Kit (EpiZyme). A total of 10 μg of proteins was separated by SDS-PAGE and then transferred onto the polyvinylidene fluoride membranes (EpiZyme). Following blocking by protein-free rapid blocking buffer (EpiZyme), the membranes were incubated with primary antibodies overnight at 4°C and further with secondary antibodies (described later and diluted according to the manufacturer’s recommendation) at room temperature for 2 h. Finally, the Omni-ECL pico light chemiluminescence kit (EpiZyme) was used for the visualization of the proteins. Immunofluorescence analysis A. castellanii cells expressing foreign protein were harvested by centrifugation at 500 × g for 5 min at room temperature. The pellets were washed with PBS twice, resuspended and fixed with 4% paraformaldehyde in PBS (Servicebio) on a slide for 20 min. The fixed cells were permeabilized in permeabilize solution (Servicebio) for 20 min and washed three times on a rocker device with PBS, for 5 min each time. The objective area of the slide was covered with 3% bovine serum albumin in PBS for 30 min. Then, cells were incubated overnight at 4 °C with antibodies (Abcam, 1: 300 diluted in PBS). The cells were washed with PBS three times, followed by incubation with FITC-conjugated goat anti-mouse PAb/ CY3-conjugated goat anti-rabbit PAb (Servicebio, 1: 600 diluted in PBS) for 50 min at RT, and then washing with PBS three times. The nuclei of cells were counterstained with DAPI (Servicebio, 500 ng/mL) for 1 min at RT in dark place. After being washed with PBS three times, the slide was covered by a glass microscope slide and then mounted with anti-fade mounting medium (Servicebio). Color profiles and co-localization were obtained using ImageJ v1.54f with the color function and the co-localization analysis, respectively.
Institutions
- Shenzhen University