IP-mass spectrometry of MeCP2 binding proteins during cell cycle re-entry

To reveal the proteins of the ubiquitination system possibly interacting with MeCP2, NIH3T3 cells were synchronized by serum starvation for 36 hours and collected following 15% FBS stimulation for 3 or 9 hours. Total protein (1 mg) was incubated with anti-Mecp2 (5 μg) overnight at 4 °C with constant mixing. IgG was used as the negative control. Then, antibody was incubated with Dynabeads for 2 h with shaking. After three washings, retained proteins were eluted using 30 μl of SDS lysis buffer. Eluted Mecp2-associated cellular proteins were separated by SDS-PAGE and stained with Coomassie blue. Trypsin was used to digest stained protein bands. An Orbitrap Elite mass spectromete was used to analyze the digested samples by Applied Protein Technology Co., Ltd. (Shanghai, China). Using Mascot as a search engine, fragment spectra were scanned against the Uniprot database to identify proteins.