Esrrb

Immunofluorescence was performed essentially as in Sridharan et al.6 (link) Antibodies used were Nanog (1:100; CosmoBio Co RCAB0002P-F), Esrrb (1:200; R&D Biosystems H6075), Pecam1 (1:100; BD Bioscience 557355).

Cells were washed with cold PBS and harvested with RIPA buffer containing complete protease inhibitor cocktail (Roche). Protein concentration was measured according to the Bradford method^{25 (link)}. Next, 50 µg of total protein was incubated at 100 °C for 10 min with electrophoresis sample buffer. The samples were loaded on each well and separated by 6–20% SDS-polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane. The blot was blocked with 5% skim milk for 1 h at room temperature and incubated overnight at 4 °C with primary antibodies against Six1 (1:1000, Cell Signaling), Eya1 (1:500, Abcam), Pax3 (1:500, R&D Systems), Esrrb (1:1000, R&D Systems), GRB2 (1:1000, Santa Cruz), and GAPDH (1:2000, Cell Signaling). After incubation with secondary antibodies (1:2000, Cell Signaling) conjugated to horseradish peroxidase, signals were detected by Super Signal West Dura Extended Duration Substrate (Pierce). The signals were visualized using an Image Analyzer (UVP) or exposed to medical X-ray film.