Figure 3: Expression analysis of EhLINE1 and EhSINE1 in the Eh-ORF2-SN cell line.

(a) Northern hybridization was performed with total cellular RNA isolated from cells doubly transfected with pEh-ORF2 and pEh-SN (Eh-ORF2-SN), or with pEh-SN and EhHYG-tetR-O-CAT vector (Eh-SN). Position of the hybridization probe in EhLINE1 is indicated in upper panel. The 2.9-kb band corresponds to the ORF2 transcript after induction with 10 μg ml⁻¹ tet. No full-length EhLINE1 transcripts of 4.8 kb are seen. (b) Western blotting followed by immunodetection with affinity-purified anti-ORF1 and anti-EN antibodies was performed using total cellular lysate from the Eh-ORF2-SN cell line. Bands of 60 kDa (ORF1p) and 111 kDa (ORF2p) are indicated. Anti-actin antibody was used for loading control. (c) The marked EhSINE1 transcript was detected by RT–PCR with total RNA. RT reaction was with primer A2 from the tag followed by PCR with primers S1 and A2 (position of primers shown in upper panel). The expected size PCR product (275 bp) was obtained. No amplicon was obtained from untransfected cells (N).