Fig. 5

Analysis of LSSL-mediated innate immunity in lamprey serum. A Determination of cell activities elicited by 30% serum, 50 µg LSSL, and 30% LSSL-depleted serum on HeLa cells after 1 h. Data are presented as the mean percentage ± SD of three independent experiments. B Native LSSL was purified from serum when the HeLa cells were not pure, and the interaction between LSSL and MASP-1 was detected. C Immunofluorescence of LSSL, MASP-1, and C3 deposited on the surface of HeLa cells treated with lamprey serum and LSSL-depleted serum. Scale bars, 10 µm. IgG was used as the control. D Histogram showing the statistics of the above-mentioned results in terms of fluorescence intensity. The data are presented as the mean ± SD. E Quantitative analysis of the proteins on the cells using Alexa 488 staining, followed by flow cytometry. F, G Histogram showing the fluorescence intensity and cell percentage statistics of the above-mentioned flow cytometry results. The data are presented as the mean ± SD. H Western blotting analysis of the expression of LSSL, MASP-1, and C3 on HeLa cells using specific antibodies. Lamprey C3 is composed of three chains of α, β, and γ. We used β and γ chains to create rabbit polyclonal antibodies. The positions are shown in the figure. I Histogram showing the statistics of the western blotting results. All experiments were replicated at least three times, and similar results were obtained (n = 3, ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). J In lampreys, LSSL recognizes glycans and activates MASP-1 to cleave C3. The active ingredient C3b recruits effector molecules to lyse pathogens