FGF13 in septic lung injury: A regulator of the ERK/aerobic glycolysis axis in the inflammatory state

HEK293T cells were transfected with Flag-FGF13 plasmids, and cells were performed with immunoprecipitation by Flag antibody or IgG (CST, 3900, USA) with protein A/G beads (Meck, LSKMAGAG10, USA). Coimmunoprecipitation was carried out in HEK293T cells and visualized with silver staining by using the beyotime fast silver stain kit (P0017S). Then mass spectrometry was used to detect all the proteins connected with Flag-FGF13. The assay was carried by Shanghai Bioprofile Technology Company Ltd. In brief, the gradient consisted of 5~35% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nL/min for 10 min, 35~100% (v/v) acetonitrile in 0.1% (v/v) formic acid at a flow rate of 200 nL/min for 2 min and 100% acetonitrile in 0.1% formic acid at a flow rate of 200 nL/min for 8 min. The eluted peptides were ionized and introduced into a Thermo Fisher LTQ Velos Pro mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) using a Proxeon nanoelectrospray ion source. Survey full-scan MS spectra (from m/z 200~1800) were acquired. A full mass spectrum (m/z 200~1800) was followed by fragmentation of the ten most abundant peaks, using 35% of the normalized collision energy for obtaining MS/MS spectra. All peptide assignments were verified by manual inspection. The information about the proteins connected with Flag-FGF13 was summarized in Supplementary materials: Proteins connect with Flag-FGF13.‘’