Figure 3: TDP1 catalytic activity (Gel-based assay) correlates with the level of TDP1 protein in SCLC cells. (A) Diagrammatic representation of TDP1 processing of a Cy5.5 labelled oligonucleotide harbouring a 3’-phosphotyrosine modification. The product of a TDP1 reaction is a Cy5.5 labelled oligonucleotide with 3’-phosphate. (B) The indicated amounts of WCE (20 ng and 25 ng) were incubated with 50 nM substrate in assay buffer at 37°C for 1 hr. Reaction products were separated on a 20% Urea SequaGel by gel electrophoresis at 190 V for 2 hr and visualised using a FujiFilm Fluor Imager FLA-5100 at 635 nm. Arrows denote the position of the substrate (PY) and cleaved product (P). (C) Quantification of the average relative TDP1 activity normalised to that obtained from HCC33 cell line from 3 independent experiments ± STD. Recombinant TDP1 (8 pM) was employed as a positive control. (D) A graphical representation of the Pearson’s correlation coefficient (r) between TDP1 protein level and activity.