iProXProteomicsZeyu CaiHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0005158000caizy@bjmu.edu.cnPeking UniversityiProXfalseIdentification of ALK3-associated proteinsThe epitope-tagging strategy to isolate ALK3-interacting protein complexes was performed. In brief, 293T cells were transfected with pcDNA3.1-FLAG-ALK3 for 48 hours. The cell pellets were resuspended in IP lysis buffer, and centrifuged at 12,000 rpm for 30 minutes at 4 °C. The cell lysates were filtered with 0.45 μm syringe filters, and then the supernatants were incubated with anti-FLAG antibody or normal IgG (negative control) overnight at 4 °C, and then incubated with Protein A/G agarose beads (sc-2003, Santa Cruz) for 6 hours at 4 °C. After that, the beads were washed with IP lysis buffer for 3 times and collected, and glycine-HCl solution (pH=3.5) was used to elute the bound polypeptides. The final eluates were subjected to an in-solution digestion and further mass spectrometry peptide sequencing.Sat Oct 08 00:00:00 GMT+01:00 2022PXD0372109606