Plgs 2

FLAG-tagged HPIP complex was obtained by immunoprecipitation with anti-Flag from 10⁸ ZR75-1 cells stably expressing FLAG-HPIP according to the manufacturer’s protocol (Sigma-Aldrich). Cells were lysed in IP buffer (20 mM tris at pH 8.0, 0.25 M NaCl, 0.5% NP-40, and 5 mM EDTA) and centrifuged to get supernatants. The supernatants were immunoprecipitated with anti-Flag agarose beads for 4 hours at 4°C. The beads were washed four times with IP buffer, and the FLAG-tagged HPIP complex was eluted with Flag peptide. The protein complex was separated by SDS-PAGE, followed by Coomassie brilliant blue staining. Differential bands were excised and subjected to mass spectrometry analysis. In-solution and in-gel digestion were performed as previously described (54 (link)). Briefly, gel bands were minced and destained, and proteins were reduced and alkylated. Trypsin digestion was performed, and digested peptides were isolated, vacuum-dried, and reconstituted. The treated samples were analyzed by nanoLC-MS/MS (nanoACQUITY UPLC and SYNAPT G2 HD mass spectrometer, Waters). Tandem mass spectrometry data were obtained with data-dependent analysis mode and analyzed using PLGS 2.4 software (Waters), and the resulting peak list was searched against the National Center for Biotechnology Information database with the MASCOT search engine.

Sediments from organoids or cells were lysed in IP buffer containing ethylenediamine tetraacetic acid and underwent IP. Agarose beads were eluted with FBN1 peptide. After SDS‐PAGE and silver‐staining, MS was conducted as previously described [28 (link)]. Subsequently, MS data were acquired from Data Dependent Analysis mode and analyzed with the PLGS 2.4 software (Waters, Shanghai, China). Finally, with the aid of MASCOT search engine, the resulting peak list was searched in the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/genome/).