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. 2022 Sep 14;13(5):e00358-22. doi: 10.1128/mbio.00358-22

FIG 3.

FIG 3

Calpain-1 mediates antiviral effects of protein fraction III. (A) Mucus protein fraction III was prepared using nondenaturing PAGE, collected through electroelution and purified using size-exclusion chromatography. Lanes 1 to 3 represent three replicates of fraction III. Shown is a chromatographic profile of fraction III in mucus obtained using an AKTA Purifier system with a SEC-150 gel-filtration column. The main peaks accounting for 74.79% of the total area were collected and verified with respect to molecular weight to obtain the purified protein fraction III. (B, C) Following incubation with the purified protein in fraction III, Vero E6 cells were inoculated with PEDV. Anti-PEDV effects were evaluated via Western blotting (B) and plaque reduction assay (C). (D) After verifying its antiviral activity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine the amino acid sequence of the purified protein in fraction III. The matched proteins, based on peptide fragment information, are shown in a bubble diagram. The protein molecular weight is shown on the x axis, the percentage of protein coverage is shown on the y axis, and the color of the dot represents the score. (E) Plaque formation ability following pretreatment with calpain-1 (0 to 10 μg) for 1 h. (F) Antiviral effect of the purified protein in fraction III treated with calpain-1 inhibitor for 1 h. (G, H) Cellular viral RNA levels for different groups (G) and viral titers (H) in medium quantified using RT-qPCR and plaque assay, respectively. (I) Purification of calpain-1 from the purified protein in fraction III using affinity chromatography. SDS-PAGE analysis of purified calpain-1. “Input” indicates the purified fraction III, “Outflow” indicates the flowthrough, “Elution” indicates the purified calpain-1. (J to L) mRNA expression (J), virus titer (K), and cellular protein (L) of PEDV in the culture supernatant of Vero E6 cells inoculated with PEDV pretreated with the input proteins (fraction III, 10 μg/mL) and the elution proteins (purified calpain-1, 10 μg/mL). The data are presented as means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; and ***, P < 0.001. BSA, bovine serum albumin; CAP, calpain-1; PEDV-N, PEDV nucleocapsid.