PMC:7376845 / 7522-8881
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7376845","sourcedb":"PMC","sourceid":"7376845","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7376845","text":"293FT cells were seeded onto 6-cm dishes 24 hours before transient transfection using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s protocol. At 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (PBS) twice. Cells were then resuspended in 2× Laemmli sample buffer, boiled and sonicated. Clarified supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13,000 rpm at 4 °C to remove the cell debris and further analysed by western blot (WB) analysis. Equal amounts of total cell lysates were loaded per lane and resolved using electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA, United States). The membrane was blocked in 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 hour at room temperature (RT) and incubated with primary antibodies at 4 °C overnight. After the membrane was washed with TBST, it was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific) at RT for 1 hour. The membrane was then washed with TBST again and bound antibodies visualised with enhanced chemiluminescence substrate (Thermo Fisher Scientific) using ChemiDoc MP Imaging System 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