Fig. 1: Screening and identification of putative membrane proteins that interact with mouse Pgk1. | Communications Biology

Fig. 1: Screening and identification of putative membrane proteins that interact with mouse Pgk1.

From: Extracellular Pgk1 interacts neural membrane protein enolase-2 to improve the neurite outgrowth of motor neurons

Fig. 1

a The pull-down assay. Membrane proteins extracted from NSC34 cells were pulled down by recombinant mouse Pgk1 fused with Flag (Pgk1-Flag) produced from Sf21 insect cells. The precipitated proteins were analyzed on SDS-PAGE by silver staining: lane 1, protein markers; lane 2, protein profiles of mixture containing Flag beads, extracts of Sf21 wild-type (WT) cells and membrane proteins extracted from NSC34 cells; and lane 3, protein profiles of mixture containing Flag beads, Pgk1-Flag produced by Sf21 and membrane proteins extracted from NSC34 cells. Location of the Pgk1-Flag band was marked with an empty arrow. Brackets on the right represented the gel slices excised for LC-MS/MS. b Three putative proteins that interacted with Pgk1-Flag were screened by crosslinking-immunoprecipitation (IP). The TRPC5-Myc, Tlr9-Myc and Eno2-Myc proteins were incubated in medium containing Pgk1-Flag, followed by cell surface crosslinking-IP using anti-Flag (IP: Flag) and then assessed by western immunoblot (IB) using either anti-Flag (Flag) or anti-Myc (Myc). Only transfection of Eno2-Myc was detected by a positive band, as marked with a solid arrow. Input represents 10% of the total cell extract used for each immunoprecipitation. c Cell surface crosslinking-IP to demonstrate the direct interaction between Pgk1-Flag and Eno2-Myc. After Eno2-Myc-expressing Sf21 cells were incubated in Pgk1-Flag-containing medium for 2 h, the cell lysate was immunoprecipitated with either anti-Flag (IP: Flag) or anti-Myc (IP: Myc), and western blot was performed using anti-Flag (Flag) or anti-Myc (Myc). Input represents 10% of the total cell extract used for each immunoprecipitation. Data were averaged from three independent experiments. d Quantification of the intensities of Flag and Myc shown on cell surface crosslinking-IP. The IP intensities of Pgk1-Flag and Eno2-Myc were individually normalized as 1. Data were averaged from three independent experiments and presented as mean ± SD (n = 3). Student’s t test was used to determine significant differences between each group (***P < 0.001). The same protein extracts from each experiment were loaded onto separate western blots and probed for individual proteins, in parallel.

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