Fig 1: IGF2 point mutation confers superior internalization and neutralization potency of AF03-IL. (A) AF03-ILm1 is prepared and characterized by SDS-page. (B) AF03-IL or AF03-ILm1 (0.3 µM) is incubated with cells at 4 °C for 1 h. PE-conjugated secondary antibody is added and the fluorescence is detected by flow cytometry. (C-F) The AF03-IL or AF03-ILm1 (0.3 µM) is incubated with HEK293T cells at 37 °C for 1-48 h to allow internalization. The fluorescence is detected by flow cytometry (C). The localization of antibodies in LE/LY is assayed by confocal microscopy (D). The supernatants are collected for analysis of antibody contents by ELISA (E). The cellular lysates are pooled for antibody detection by western blotting (F). (G) Pseudotyped ebolavirus species are incubated with AF03-IL or AF03-ILm1 respectively, and then infects HEK293T cells. Luciferase is assayed and inhibition rates are calculated. Representative plots or images were shown. The data were pooled from at least three repeated experiments with similar results. Data are presented as mean±SD. Statistical significance was determined using the Two-way ANOVA. *p<0.05, ***p<0.001, ****p<0.0001.
Fig 2: AF03-IL shows fine binding potency to Marburg virus (MARV) glycoprotein (GP) as well as IGF2R segments. (A,B) The binding kinetics of AF-03 (A) and AF03-IL (B) to MARV GP are detected by SPR assay. (C,D) The binding kinetics of AF03-IL (C) and IGF2 (D) to domain 11–13 of IGF2R protein are detected by SPR assay. The data were representative of three repeated experiments with similar results. Data are presented as mean ± SD.
Fig 3: The internalization and neutralization activity of AF03-IL are impaired by IGF2-IGF1R interaction. (A) The surface expression of IGF1R and IGF2R on HEK293T cells was detected by flow cytometry. (B) AF03-IL (0.3 µM) was incubated with IGF1R-deficient or IGF1R-sufficient cells at 4 °C for 1 h. A PE-conjugated secondary antibody was added and fluorescence intensity was measured by flow cytometry. (C-F) AF03-IL (0.3 µM) was incubated with IGF1R-deficient or IGF1R-sufficient cells at 37 °C for 1-48 h to allow internalization. Fluorescence was detected by flow cytometry (C). Antibodies localization in LE/LY compartments was assayed by confocal microscopy (D). Supernatants were collected for quantification of antibody levels by ELISA (E). Cellular lysates were pooled for antibody detection by Western blotting (F). (G) AF03-IL was mixed with recombinant human IGF1R protein or control protein (CP), then incubated with pseudotyped ebolavirus species, followed by infecting HEK293T cells. Luciferase is assayed and inhibition rates are calculated. Representative plots or images were shown. The data were pooled from at least three repeated experiments with similar results. Data are presented as mean±SD. Statistical significance was determined using the Two-way ANOVA. *p<0.05, **p<0.01, ***p<0.001.
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