![Figure 2. Interaction of KRS with syntenin for secretion. (A) HCT116 cells were incubated in the starvation (Starv.) condition in the absence and presence of TNF-α, and the interaction of KRS with syntenin was determined by coimmunoprecipitation with an anti-syntenin antibody. (B) The cellular interaction of KRS and syntenin was determined by BiFC. HCT116 cells were cotransfected with Flag-KRS fused to VN173 (Venus N-terminal domain) and HA-syntenin fused to VC155 (Venus C-terminal domain). The cells were incubated in normal and starvation conditions for 6 h, and reconstitution of Venus green fluorescence was shown by fluorescence microscopy. Expression of KRS was observed by red fluorescence signal from Alexa Fluor 594–conjugated anti-FLAG antibody (60×). Nuclei were visualized by DAPI staining (blue). (C) Secreted KRS and syntenin (syn) were detected by immunoblotting of proteins from HCT116 cells treated with different amounts of siRNA against syntenin. The cells were incubated in starvation conditions for 24 h. Secreted proteins were precipitated with TCA, and the amounts of KRS were determined by immunoblotting. (D) The levels of KRS were also determined, as described in Fig. 1 B, in isolated exosomes and cell lysates of HCT116 cells transfected with siRNA against syntenin. (E) Interaction of KRS-Myc (WT and ΔC5 [C-terminal 5-aa–deleted mutant]) with syntenin was determined by coimmunoprecipitation from HCT116 cells after 1-h incubation in the starvation condition. Syntenin was precipitated from whole-cell lysates (WCLs) with its specific antibody, and the coprecipitated KRS was immunoblotted with anti-Myc antibody. IP, immunoprecipitation (F) Secretion of KRS (WT and ΔC5) from HCT116 cells was determined in the 24-h starvation media as in C. (G) The amounts of KRS WT and ΔC5 were determined by immunoblotting in the exosomes isolated from HCT116 cells incubated in the starvation condition as in Fig. 1 B. (H) KRS mutant in which the last four aa’s, GTSV, were changed to alanine (C4A) was constructed. KRS WT and the C4A mutant secretion from HCT116 cells starved for 24 h, as described in Fig. 1 B, was compared.](https://cdn.rupress.org/rup/content_public/journal/jcb/216/7/10.1083_jcb.201605118/8/jcb_201605118_fig2.jpeg?Expires=1771704457&Signature=IhoIzgAkm6XAkKMcGlGL5K5KKeFAafsorMKjTzoxj7WjcwP6o~nE~CuV7rlRXfLJVZmfuj8dxj7yoVKRk~zORngu5P5u5oG9Eep3TEfgB-ZcDI00Yb7b9G6e0yhpWabzSnsvfQGrlfaXLY3PvqHnQjnb9xQK-EGkQUoxIvL7BSEyvYcjT6IzQy-Il4uid1z9Q~4J8bwIQCHHCK7QpXBvWSb6SqGPnXl0h0SQvvi-Tg94fA90Z71D32AK0jD48xrS-8XxXq4uGObC305ND-kDiH4QRrvhOw-aXmVnDKceDPsS8rkImy3JCWopBVaqIW8i5DdTWn9c2pnBpVygUkSs8A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Interaction of KRS with syntenin for secretion. (A) HCT116 cells were incubated in the starvation (Starv.) condition in the absence and presence of TNF-α, and the interaction of KRS with syntenin was determined by coimmunoprecipitation with an anti-syntenin antibody. (B) The cellular interaction of KRS and syntenin was determined by BiFC. HCT116 cells were cotransfected with Flag-KRS fused to VN173 (Venus N-terminal domain) and HA-syntenin fused to VC155 (Venus C-terminal domain). The cells were incubated in normal and starvation conditions for 6 h, and reconstitution of Venus green fluorescence was shown by fluorescence microscopy. Expression of KRS was observed by red fluorescence signal from Alexa Fluor 594–conjugated anti-FLAG antibody (60×). Nuclei were visualized by DAPI staining (blue). (C) Secreted KRS and syntenin (syn) were detected by immunoblotting of proteins from HCT116 cells treated with different amounts of siRNA against syntenin. The cells were incubated in starvation conditions for 24 h. Secreted proteins were precipitated with TCA, and the amounts of KRS were determined by immunoblotting. (D) The levels of KRS were also determined, as described in Fig. 1 B, in isolated exosomes and cell lysates of HCT116 cells transfected with siRNA against syntenin. (E) Interaction of KRS-Myc (WT and ΔC5 [C-terminal 5-aa–deleted mutant]) with syntenin was determined by coimmunoprecipitation from HCT116 cells after 1-h incubation in the starvation condition. Syntenin was precipitated from whole-cell lysates (WCLs) with its specific antibody, and the coprecipitated KRS was immunoblotted with anti-Myc antibody. IP, immunoprecipitation (F) Secretion of KRS (WT and ΔC5) from HCT116 cells was determined in the 24-h starvation media as in C. (G) The amounts of KRS WT and ΔC5 were determined by immunoblotting in the exosomes isolated from HCT116 cells incubated in the starvation condition as in Fig. 1 B. (H) KRS mutant in which the last four aa’s, GTSV, were changed to alanine (C4A) was constructed. KRS WT and the C4A mutant secretion from HCT116 cells starved for 24 h, as described in Fig. 1 B, was compared.
