Fig. 5: SLC1A5 was identified as the receptor of P-LPK.

a P-LPK-specific binding proteins were investigated via Co-immunoprecipitation test. 50 μg/ml Biotin-P-CON (P1) or Biotin-P-LPK (P2) was incubated with HCT116 at 37 °C for 2 h. Then cells were lysed and centrifuged at 12,000 rpm for 20 min at 4 °C. Differential protein bands between P1 and P2 were indicated by the red dashed box. (Details reference to materials and methods). S, supernatant group; S1, supernatant group 1; S2, supernatant group 2; co-IP products, the beads-P-LPK-specific binding protein compounds. b Targeted proteins were detected by mass analysis and several closely related P-LPK-specific binding proteins were focused. c The 3D structure models of the P-LPK peptide and SLC1A5. Molecular interactions of P-LPK to SLC1A5 were predicted by molecular docking. d–f The binding pocket of SLC1A5 was represented by (d) the electrostatic potential, where P-LPK were shown in pale blue stick (e) ribbon, where P-LPK was shown in cyan (f) 2D topological binding mode by Ligplot + (v2.2), in which hydrogen bonds were shown in green dotted lines and hydrophobic interaction were shown in red edges. g The affinity of P-LPK for SLC1A5 was measured by surface plasmon resonance (SPR) and expressed as resonance units (RU). h Co-localization of SLC1A5 (Red) and P-LPK (Green) was observed by dual immunostaining. Bar,10 μm. i P-LPK was mutated (Sequence APATVSSDMSLA) and the fluorescence intensity of FITC labeled mutant peptide (FITC-P-MUT) bound to HCT116 cells. Bar,10 μm.