Protein a g magnetic beads

The full‐length open reading frame of FGF4 (1–352 aa) and the IG2 and IG3 domain regions (81–431 aa) of FGFR2 were respectively cloned and inserted into the pcDNA3.1‐EGFP and pcDNA3.1–3 × flag expression vector (Invitrogen) to construct the following plasmids: pcDNA‐flag‐FGF4, pcDNA‐flag‐FGFR2, and pcDNA‐EGFP‐FGFR2. The primers used to amplify the gene fragments were listed in Table 1. For the coimmunoprecipitation (Co‐IP) assays, HEK‐293T cells were seeded in a T25 flask, incubated overnight, and then transfected with the indicated expression plasmids (pcDNA‐flag‐FGF4 + pcDNA‐EGFP‐FGFR2, pcDNA‐flag‐FGF4 + pcDNA‐EGFP, pcDNA‐EGFP‐FGFR2 + pcDNA‐flag) using Lipo8000 transfection reagent (C0533FT, Beyotime). At 48 h post‐transfection, the cells were washed with precooled PBS and lysed with IP cell lysis buffer containing protease inhibitor PMSF (P0013, Beyotime). After centrifugation at 13,000g for 20 min at 4°C, the supernatants of the cell lysates were immunoprecipitated using Protein A/G magnetic beads (P2055, Beyotime) which were bound with anti‐flag (M20008, Abmart) or anti‐GFP antibodies (AG281, Beyotime) and rotated slowly at 4°C overnight. The precipitates were washed 10 times with lysis buffer and then eluted by boiling the pellets with 5 × SDS PAGE loading buffer. Finally, the eluted samples were analyzed by immunoblotting with the indicated antibodies.

RIPA lysis (1 ml) were added to 10 cm dish with cells and proteins were extracted following western blot methods. After incubation with 2 µg antibody for 2 h at 4°C, 30 µl protein A/G magnetic beads (Beyotime) was added and inverted overnight. Next day, samples were washed thoroughly with RIPA lysis buffer (Beyotime) three times, incubated in 30 µl of 2×SDS-PAGE sample loading buffer (Beyotime) and boiled at 100°C for 10 min for subsequent western blot experiments.

The protein level of GAPDH, TCF-1, β-catenin, JunB and NFATc2 in ILC3s was detected by Western blot with the following antibodies: GAPDH (Beyotime, AF2819), TCF-1 (clone C63D9, Cell Signaling Technology, 2203), β-catenin (BD, 610154), JunB (clone C37F9, Thermo Fisher Scientific, PA1-835) and NFATc2 (clone 25A10.D6.D2, Thermo Fisher Scientific, MA1-025). In co-immunoprecipitation (co-IP) experiments, ILC3 pellets were resuspended in cell lysis buffer (Beyotime, P0013) including 1 mM PMSF (Beyotime, ST506). Then protein A + G magnetic beads (Beyotime, P2108), mouse IgG1 isotype control (clone G3A1, Cell Signaling Technology, 5415) and mouse anti-β-catenin (clone D10A8, Cell Signaling Technology, 8480) were used for IP. The IgG1-binding and β-catenin-binding proteins were further identified by Western blot.

Co-IP was used to demonstrate protein-protein interactions through binding interactions. Mass Spectrometry was applied to identify the specific proteins interacting with MFSD4A. Briefly, we incubated anti-flag, anti-GFP, anti-HA, or anti-IgG antibodies with cell lysates overnight at 4 °C. Then, washed protein A/G magnetic beads (Beyotime) were added to the cell lysate on the mixer to adsorb the immune complexes at room temperature for 1 h. Immune complexes were eluted from the magnetic beads, denatured by boiling, separated using SDS-PAGE, and then subjected to mass spectrometry or western blotting assays.