PMC:4925210 / 43876-45019 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4925210","sourcedb":"PMC","sourceid":"4925210","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4925210","text":"Cell surface biotinylation\nWild type, Pkd1−/− MEFs, or HEK293T cells co-transfected with TRPP2 and wild type PKD1 (PKD1WT) or PKD1S99I were washed 3 times in PBS (pH 8.0), scraped, and re-suspended in 1 ml PBS (pH 8.0). Cell surface proteins were labelled with 1 mM EZ-Link NHS-PEG4-biotin (Thermo Scientific, 21329) for 30 min at room temperature. Biotinylation was terminated by 3 washes with PBS containing 100 mM Glycine, pH 3.0, and lysed in 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl at pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5% NP-40, and 10% sucrose with protease inhibitor cocktail (Roche Applied Science) at 4°C for 30 min. Lysates were collected by centrifugation (18,000 × g, 20 min) and biotin-labelled surface proteins were captured on streptavidin–agarose beads at 4°C by an overnight incubation. Proteins bound to beads were collected by a brief (1 min) centrifugation, and pellets were washed 3 times with lysis buffer for 20 min at 4°C. Biotin-labeled surface proteins were eluted with SDS gel-loading buffer (50 mM Tris-HCl at pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, and 10% glycerol) and analyzed by western blotting.","divisions":[{"label":"Title","span":{"begin":0,"end":26}}],"tracks":[]}