Expression of EmIR1 and EmIR2 in E. multilocularis larval stages. A) Semi-quantitative RT-PCR of emir1 and emir2 expression. Serial 1/10 dilutions of cDNA from metacestode vesicles (MC), primary cells (PC) as well as dormant (PS-) and low pH/pepsin-activated protoscoleces (PS+) were subjected to gene-specific PCR using intron-flanking primers. PCR products were separated on a 1% agarose gel and stained with ethidium bromide. The constitutively expressed gene elp was used as control. B) Western blot and immunoprecipitation employing the EmIR1 anti-serum. EmIR1 was immunoprecipitated from metacestode vesicles and treated with β-mercaptoethanol (beta-MeOH) at concentrations of 0%, 1% and 10%. Probes were then separated on a 12.5% polyacrylamide gel and developed using the anti-EmIR1 antiserum. ‘pro’ and ‘beta’ indicate the pro-form and the β-subunit of EmIR, respectively. C) Immunoprecipitation and Western blot using the anti-EmIR2 serum. EmIR2 was immunoprecipitated from protoscolex preparations, samples were then supplemented with 1% or 10% of β-mercaptoethanol (β-ME) and separated on a 10% SDS gel. Western blot was carried out using the anti-EmIR2 antiserum. D) Immunodetection of EmIR1 and EmIR2 in different larval stages using immune sera. Parasite larvae were lysed, protein preparations were then separated by SDS-PAGE, blotted onto a membrane and detected by the antisera. The purified anti-EmIR2 immune serum recognized the EmIR2 β-subunit at 87 kDa and a second band at 60 kDa. The EmIR1 β-subunit was detected at 150 kDa using the anti-EmIR1 immune serum. Actin was used as a loading control. Mc, metacestode vesicles; Pc, primary cells; Ps-, dormant protoscoleces; Ps+, activated protoscoleces.
Hemer et al. BMC Biology 2014 12:5 doi:10.1186/1741-7007-12-5