PMC:3432770 / 48541-50552
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3432770","sourcedb":"PMC","sourceid":"3432770","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3432770","text":"Immunoprecipitations, Western blots, and proteasome inhibition\nImmunoprecipitations and Western blots in renal cells were performed as described previously (Delous et al., 2009). In brief, cells were lysed for 10 min with lysis buffer (1% Triton X-100, 50 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 1 mm sodium orthovanadate with complete protease inhibitor cocktail; Roche). Insoluble debris was removed by centrifugation at 14,000 g for 15 min. Proteins were detected with mouse anti-Myc (1:1,000; Santa-Cruz Biotechnology, Inc.), mouse anti-Flag (1:1,000; Sigma-Aldrich), rabbit anti-Flag (1:1,000; Sigma-Aldrich), mouse anti-V5 (1:1,000; AbD Serotec), mouse anti-GFP (1:1,000; Roche), mouse anti–α-tubulin (1:5:000; Sigma-Aldrich), rabbit anti-Dvl2 and anti-Dvl3 (1:1,000 and 1:500, respectively; Cell Signaling Technology), and rabbit anti-NPHP3 (this paper). Images were captured with a Fusion Fx7 system and quantified with Bio-1D software (Vilbert-Lourmat). To inhibit proteasome-dependent degradation in MDCK cells, cells grown for 8 d were treated overnight with 10 µm of clasto-lactacystin β-lactone (Sigma-Aldrich) before collection. Cells were harvested and lysed as described above. For Western blots with zebrafish embryos, pools of 15 embryos injected with Dvl2-Myc RNA ± Mo-Rpgrip1l were collected for Western blot analysis. Embryos were manually dechorionated. For 80% epiboly embryos, a hole was made with forceps into the yolk sac to remove the vitellus. Older embryos were transferred to 200 µl of deyolking buffer (55 mM NaCl, 1.8 mM KCl, and 1.25 mM NaHCO) by pipetting with a 200-µl tip to disrupt the yolk sac. Embryos were shaken 5 min at 1,100 rpm to dissolve the yolk. Cells were pelleted for 2 min at 1,800 rpm, resuspended in Laemmli buffer, and stored at −80°C (Link et al., 2006). The Myc epitope was detected by 9B11 monoclonal antibody (catalog no. 2276, 1:1,000; Cell Signaling Technology). α-Actin was used as a loading control (monoclonal antibody A4700, 1:1,000; Sigma-Aldrich).","divisions":[{"label":"Title","span":{"begin":0,"end":62}}],"tracks":[{"project":"2_test","denotations":[{"id":"22927466-19755384-56740352","span":{"begin":172,"end":176},"obj":"19755384"},{"id":"22927466-16412219-56740353","span":{"begin":1801,"end":1805},"obj":"16412219"}],"attributes":[{"subj":"22927466-19755384-56740352","pred":"source","obj":"2_test"},{"subj":"22927466-16412219-56740353","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#d0ec93","default":true}]}]}}