Western blot and qPCR analysis of TsAP protein and mRNA expression levels at diverseT. spiralisstages. A SDS-PAGE analysis of crude proteins of diverse T. spiralis stages. Lane M: protein markers; Lane 1: ML; Lane 2: IIL; Lane 3: AW; Lane 4: NBL; Lane 5: rTsAP. B Identification of native TsAP in crude proteins of T. spiralis ML (lane 1), IIL (lane 2), AW (lane 3) and NBL (lane 4), and rTsAP (lane 5) by western blot analysis with anti-rTsAP serum. C Quantitative analysis of TsAP expression levels in crude proteins of diverse T. spiralis stages (ML, IIL, AW and NBL) was determined by western blot with 1:100 dilutions of anti-rTsAP serum. The graph shows the relative TsAP protein expression levels determined by densitometry from three independent experiments. *P < 0.01 compared with ML and NBL group. D qPCR analysis of TsAP mRNA transcription level at diverse T. spiralis stages. The TsAP mRNA from ML, IIL, 3-day AW and NBL were isolated and amplified by qPCR. The TsAP transcription level was calculated according to the Ct (2-ΔΔCt) method. The fold change of TsAP genes normalized to GAPDH served as an internal control gene. Three repeats for each sample were performed. * P < 0.01 compared to muscle larvae stage.