Goat n 19

Exponentially growing cells in duplicate wells of a 6-well plate were co-exposed to 1 µM gemcitabine, to activate ATR, with increasing concentrations of VE-821 for 1 h. 10 mM HU was used as a positive control as it is known to activate ATR [9 (link)]. VE-821 stock was diluted in DMSO at 200× the final concentration before diluting 1 in 200 in medium and 0.5% DMSO was used as a vehicle control. Lysates were run on 18-well Criterion gels to allow multiple samples to be run at the same time (Bio-Rad, Watford, UK). ATR activity was measured by CHK1 phosphorylation by Western blotting. Antibodies to pCHK1^Ser345 (rabbit 133D3, 1/300 Cell Signaling, Danvers, MA, USA), ATR (goat N-19, 1/300, Santa Cruz Biotechnology, Dallas, TX, USA) and actin (mouse AC-40, 1/1000, Sigma Aldrich) and the appropriate HRP-conjugated secondary antibodies (antigoat HRP, 1/2000–Santa Cruz Biotechnology, Dallas, TX, USA, antirabbit HRP 1/1000 and antimouse HRP (1/2000–Dako UK Ltd., Ely, UK) were used. Protein expression was measured by chemiluminescence from exposure to ECL Prime detection reagent (GE Healthcare, Pittsburg, PA, USA) using a G-box (Syngene, Cambridge, UK). Densitometry of bands was carried out using Genetools software (Syngene, Cambridge, UK).