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. 2012 Jun 22;40(17):8309–8324. doi: 10.1093/nar/gks591

Figure 2.

Figure 2.

Characterization of the TopBP1 interaction with RecQL4. (A) Identification of the RecQL4_N54 BD of TopBP1 by GST pull-down. Pull-down experiments were performed as in Figure 1C–D with GST-fusion constructs as bait for in vitro translated proteins as indicated. Only TopBP1 (a.a. 1233–1522) shows efficient binding to GST-RecQL4_N54. Purified GST and GST-TopBP1 (a.a. 1233–1264) stained with Coomassie brilliant blue R250 are shown on the right. (B) Real-time in vitro SPR binding analysis of RecQL4_N54 to anti-GST antibody captured GST-TopBP1 (a.a. 1233–1522). Sensorgrams of 200, 100, 50, 25, 12.5, 6.25 and 3.13 µM RecQL4_N54 binding injected in triplicate (black lines) are shown overlaid with the best fit derived from a 1:1 interaction model (red lines) (left). Fit of the equilibrium data for TopBP1 (a.a. 1233–1522) binding (middle). The purified GST-TopBP1 (a.a. 1233–1593) used for the experiment stained with Coomassie brilliant blue R250 is shown on the right.