Figure 4: TDP1 catalytic activity (Fluorescence assay) correlates with the level of TDP1 protein in SCLC cells. (A) A Metal ion sensor (indicated S) along the phosphate backbone of the substrate oligomer keeps the bound FITC-conjugated tyrosine in a non-fluorescent state (OFF). During a TDP1 driven reaction, the FITC conjugated tyrosine is released and distance generated between the released FITC-tyrosine and the oligonucleotide increases, allowing for FITC fluorescence (ON) that can be detected and quantified using a fluorescence imager. (B) A 15 μl final volume reaction containing 0.5 μg or 1.0 μg WCE from the indicated DT40 cell lines and 10 nM substrate was incubated for 10 min at room temperature. Reactions were stopped by addition of a reaction quencher and fluorescence intensity was measured using a BMG Labtech Pherastar plate reader at excitation and emission wavelengths of λex 490 nm and λem 520 nm. Fluorescence was normalised to that obtained for recombinant human TDP1 (6.5 pM) and data represent the average of 3 independent experiments ± STD. (C) Whole cell extracts (40 μg) from the indicated DT40 cell lines were separated by 10% SDS-PAGE and immunblotted using antibodies against TDP1 and actin. (D) A 15 μl final volume reaction containing 0.5 μg SCLC WCE and 10 nM substrate was incubated for 10 min at room temperature. Reactions were stopped by addition of a reaction quencher and fluorescence intensity was measured using a BMG Labtech Pherastar plate reader at excitation and emission wavelengths of λex 490 nm and λem 520 nm. Average fluorescence was normalised to that obtained for recombinant human TDP1 (6.5 pM), and data represent the average of 3 independent experiments ± STD. (E) A graphical representation of the Pearson’s correlation coefficient (r) between SCLC TDP1 protein level and TDP1 activity from 0.5 μg WCE.