Fig. 2

Protein complex purification reveals the binding partners of EDAR. A A schematic shows the workflow of EDAR complex purification. B Immunoblotting shows gel-filtration profiles in the cytoplasmic protein extracts from EDAR⁺ cells. The red rectangle indicates fractions collected for immunoprecipitation (IP) and mass spectral (MS) analysis. C Key components of EDAR complexes determined by MS. The numbers (#) of total and unique peptides (pept) of indicated proteins are shown. D GFP-IP with cytoplasmic protein extracts was followed by immunoblotting with the indicated antibodies. 5% cytoplasmic extracts from EDAR-GFP⁺ cells used as input. IP with TNFR1 antibody as the negative control. E Quantification of the levels of EDAR-associated proteins in D. The protein levels with no EDA treatment were normalized to 1.0. Data from 3 independent experiments. F Immunoblotting shows the levels of indicated proteins in Cyt or PM fraction with (+) or without (−) EDA treatment. Error bars indicate mean ± SD. *P < 0.05, **P < 0.01; Student’s t-test