Molecules and Cells

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Fig. 1.

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Fig. 1. (A) Construction of expression vectors encoding IL-15 or IL-15Rα. In the expression vectors, the natural signal peptide sequence (SS) of IL-15 or IL-15Rα were replaced with that of IL-2. TM, transmembrane domain; CY, cytoplasmic domain. (B) Quantitation of IL-15 or IL-15:IL-15Rα complex, secreted from 1 × 106 cells of transfected clones for 24 h incubation by ELISA. The monoclonal antibody used to detect IL-15 (DuoSet ELISA kit; R&D System) did not bind to the IL-15:IL-15Rα complex. The monoclonal antibody to the IL-15:IL-15Rα complex (Platinum ELISA; Affymetrix) did not bind to IL-15 alone. (C) Immunoprecipitation (IP) of IL-15 and IL-15Rα from the culture supernatants of the indicated transfectants. The amount of immunoprecipitated proteins were analyzed by immunoblotting (IB) with the indicated antibodies. Then, the relative intensive was shown in the bar graph. (D) Transfected cell proliferation analysis in vitro by MTT assay. The cell proliferation was correlated with the absorbance at optical density 570 nm. (E) Secreted exogenous IL-15 and IL-15:IL-15Rα complexes stimulated splenocyte proliferation in vitro. Culture supernatants from 1 × 106 cells of each transfected tumor clone were collected after 24 h. The splenocytes from normal BALB/c mice were re-suspended in the medium containing the indicate culture supernatants for 72 h. Then, the proliferation of splenocytes was assayed by MTT assay. Data are mean ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
Mol. Cells 2019;42:869~883
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