Anti flag affinity gel

Immunoprecipitation (IP) and mass spectrometry assay were conducted to investigate the interacting proteins of peptide MBOP. Three 100 mm dishes of cells were transfected with plasmids pcDNA3.1+, pORF-FLAG, and pORFmut-FLAG at a confluency of 50–60%. After 48 h, cells were harvested with Pierce™ IP Lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with Halt™ Protease Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA), and anti-FLAG affinity gel (Yeasen, Shanghai, China) was used to perform IP assay, then 12% SDS-PAGE was conducted to separate the interacting proteins. Next, protein bands with the most apparent discrepancies were cut to perform further mass spectrometry assay (Applied Protein Technology, Shanghai, China) and KEGG analysis.

We stably expressed vector control, HA-Flag-TMEM43 WT and HA-Flag-TMEM43 S358L mutant in A549 cells with lentivirus and selected the positive infected cells with blasticidin (BSD) treatment for 7 days. Three cell lines were lysed, immunoprecipitated (IP) with Flag antibody (Anti-Flag Affinity Gel, YEASEN, Cat# 20585ES08) and then HA antibody, the obtained products were performed trypsin digestion and mass spectrometry (MS) analysis. The products of every step during IP were collected and subjected to immunoblotting with Flag antibody (Abmart, Cat# M20008) to control the quality of IP. The obtained results from MS were subjected to searching and comparison in protein database bank (PDB). MS scores referred the general abundance of specific proteins detected.

To identify the receptor of UGRP1, purified FLAG‐UGRP1 protein (5 μg) was incubated with the lysate of PEMs for 2 h at 4°C in the lysis buffer (20 mM Tris–HCl, 1% (v/v) NP‐40, 137 mM NaCl, 10% (v/v) glycerol, 2 mM EDTA and protease inhibitor cocktail, pH 7.5), followed by incubated with the Anti‐FLAG Affinity Gel (YEASEN) for 2 h at 4°C. After three washes and elution with the FLAG peptides, the proteins were used for the mass spectrometry analysis.