Figure 5

Antiserum preparation and Western blot. Three peptides, the wild-type RTEL protein, the RTEL isoform generated from splice variant 1 and RTEL767-1060 were over-expressed in E. coli. RTEL767-1060 was purified and used to prepare the antiserum for West analysis. (A) Schematic representation of the regions of RTEL that expressed in E. coli. BL21-Rosetta. RTEL-WT represents the complete coding region of RTEL; RTEL SV-1 represents the coding region of splice variants 1; RTEL767-1060 represents a shared coding regionin all potential isoform of RTEL. The roman numbers and 'PIP'specify the conserved helicase motifs and PIP box. (B). Over-expression of wild-type RTEL (WT), RTEL SV-1 (SV-1) and RTEL767-1060 (767–1060) in E. coli BL21-Rosetta induced by IPTG (1 mM). After inducement, the his-tagged recombinant proteins were over expressed. BI and AI listed below means 'before inducement' and 'after inducement' respectively. The empty pET30a-E. coli and wild-type BL21-Rosetta were used as negative controls. (C). Purification of his-tagged RTEL767-1060. RTEL767-1060 is a region shared by all potential RTEL isoforms and was used for antiserum preparation. The homogeneous of purified RTEL767-1060 is >95%. (D). Western analysis of RTEL expression. The lysates of E. coli cells that expressed wild-type RTEL and SV-1 RTEL were used as positive controls. Two bands were detected in the RTEL-expressing tissues, after comparing to the molecular marker and sizes of positive controls, the 140 kDa and the 120 kDa bands are corresponding to the wild-type RTEL and the SV-1 isoform of RTEL respectively.