Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection

Figure 2

Molecular weight of duck IL-22 and its expression in normal tissues and stimulated splenic lymphocytes. (a) Western blot analysis to determine molecular weight of duck IL-22 protein from COS-7 cells transfected with duIL-22-MYC construct. Supernatants and cell lysates of COS-7 cells were obtained at 48 h after transfection and deglycosylated using PNGase F (100 U peptide-N-glycosidase F) at 37°C for 1 h. Supernatants and cell lysates under reducing conditions were separated using SDS-PAGE. An anti-MYC antibody was used to detect the specific bands of duck IL-22. The asterisk () and arrow indicated the normal bands and the deglycosylated bands of duck IL-22, respectively. (b) Distribution of IL-22 transcripts in normal healthy duck tissues. Total RNA was obtained from tissues of two-week-old normal ducks () and used to qRT-PCR analysis. Expression levels of β-actin were used for normalization of IL-22 expression levels, which were calibrated to the lowest expression level detected. Results are presented as the values from 2 independent experiments. (c) Duck IL-22 expression levels in mitogen-treated splenic lymphocytes. The lymphocytes were obtained from two-week-old normal ducks using Ficoll density gradient centrifugation. They were activated using 25 μg/ml poly I:C, 10 μg/m LPS, or 10 μg/ml ConA for the indicated times. Expression levels of β-actin gene were used for normalization of IL-22 expression level, which were calibrated with untreated cultured splenic lymphocytes (NC). Results are presented as the values from 2 independent experiments performed in triplicate. and .

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