Figure 4

TOP1 from HeLa cells interacts with GST-BTBD1 and GST-BTBD2.A. TOP1 Western blot. HeLa cell nuclear extracts were incubated with GST or GST-BTBD2 beads. After washing, the beads were solubilized in SDS-PAGE sample buffer and subjected to SDS-PAGE and Western blotting with TOP1 antiserum. B. Coomassie Blue stained gel showing the quantity of GST and GST fusion proteins added to the binding reactions (*). METHODS: GST-BTBD1 or GST-BTBD2 was expressed and purified from E. coli extracts. ~5 μg of GST or the GST-fusion proteins were mixed with HeLa cell nuclear extracts and incubated for 2 hr at 4°C (0.25 M NaCl). The bead pellets were then washed 3 times with ice cold PBS before solubilization in SDS sample buffer. One tenth of the nuclear extract used in the binding reaction was loaded in the lanes labeled "HeLa extract". Arrow shows co-precipitated TOP1.