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. 2016 Sep 22;6:34013. doi: 10.1038/srep34013

Figure 4. SmedOB1 is required for telomeric DNA-protein complex association.

Figure 4

(A) Nuclear extracts (NE) were obtained from si-GFP and si-SmedOB1 planarian cells. EMSA assay using indicated amounts of NE and 1 nM (TTAGGG)8 probe (lanes 2–7). Lane 1, positive control provided with the kit; lane 8, 12 nM purified human POT1 (final concentration), two arrows indicated shifted bands (upper and middle) and the lower arrow indicated the free probe. (B) The C terminal SFB tagged GFP, human POT1 and SmedOB1 were expressed in 293T cells and purified with SBP tag. Coomassie Blue staining of purified GFP-SFB, human POT1-SFB and SmedOB1-SFB. BSA proteins were used as controls. (C) Electrophoresis mobility shift Assay (EMSA) for the full-length SmedOB1 with 1 nM (TTAGGG)8 ssDNA probe. Lane 1: 200 nM GFP-SFB; Lanes 2–4: SmedOB1-SFB (50 nM–200 nM); Lane 5: SmedPOT1-SFB (200 nM) plus FLAG antibody; Lanes 6–8: human POT1-SFB (50 nM–200 nM); Lane 9: human POT1-SFB (200 nM) plus FLAG antibody. Human POT1 was used as a positive control, and GFP was used as negative control. All the amounts refer to final concentration. The upper arrow indicated the supershift, the middle arrow indicated the Protein-ssDNA shift and the lower arrow indicated the free probe. (D) EMSA for the full-length SmedOB1 with 1 nM (TTAGGG)8 telomere probe (Lanes 1–5) or (TTAGCC)8 mutant probe (Lanes 6–10). Lanes 1 and 6, no protein; Lanes 2 and 7: 200 nM GFP-SFB; Lanes 3–5, 50 nM, 100 nM and 200 nM SmedOB1-SFB (OB1-SFB).