Fig. 5

Recombinant GST- zLPTS protein interacts with zTERT. (A) Scheme of zebrafish TERT protein structures. Three fragments that corresponding to the N-terminal (zTERT-N), RNA-binding domain (zTERT-M), RT and C-terminal (zTERT-C) of full-length protein were indicated. Motifs of CP, QFP, T, 1 and 2 were further mapped in RNA-binding domain. (B) 10% SDS-PAGE analysis of recombinant GST-zLPTS fusion protein. Lane1, Cell lysate of E. coli BL21(DE3) containing the pGEX-4T2-zLPTS plasmid. Lane2, Cell lysate of IPTG-induced E. coli BL21(DE3) containing the pGEX-4T2-zLPTS plasmid. Lane3, GST-zLPTS fusion protein purified by affinity chromatography. Lane 4, the GST protein purified from the cells harboring the pGEX-4T2 vector that used as a control. The GST-zLPTS or GST protein was indicated by arrow. (C) Western blotting analysis of the in vitro translated three N-terminal Myc-tagged zTERT protein fragments (zTERT-N, zTERT-M and zTERT-C) by using anti-myc antibody. (D) Interactions between zLPTS and zTERT protein fragments by GST pulldown assay. GST or GST-zLPTS fusion protein was used to incubate with Myc-tagged zTERT-N, M or C protein fragment. Western blotting was performed with anti-myc antibody. (E) Interactions between zLPTS and zTERT-M sub-fragments by GST pulldown assay. The in vitro translated six N-terminal Myc-tagged zTERT-M protein fragments including zTERT-M, M1, M2, M3, M1 + M2 and M2 + M3 as indicated in (A) were used to incubate with GST or GST-zLPTS fusion protein. Interaction result was examined with anti-myc antibody.