Immunoprecipitation and Mass Spectrometry Identification

Immunoprecipitation was performed using PureProteome Protein G Magnetic Beads (Merck Millipore, Darmstadt, Germany) and the capture antibody, goat anti-rat IgG Fc (ab97086, Abcam), according to the manufacturer’s instructions. The bound immune complexes were analyzed by SDS-PAGE under reducing conditions. Protein bands were visualized by silver staining. Individual stained bands were excised from gels, destained, and subjected to enzyme digestion as described previously^{25 (link)}. The peptides were separated using a trypsin/lysyl endopeptidase solution (150 ng/30 μL), and amino acid sequences were determined by mass spectrometry (Easy-nLC 1000/Orbitrap Elite, Thermo Fisher Scientific). Mass spectrometry data were processed and subjected to database searches using the Mascot server (Matrix Science, Boston, MA, USA) or Sequest HT (Thermo Fisher Scientific).