GST pull-down assays

Cells grown in 10 cm dishes were lysed in 600 μl cold lysis buffer for 10 minutes prior to centrifugation at 14,000 × g for 5 minutes. The supernatant was collected and 300 μl was isolated for addition of normalised amounts of recombinant GST-tagged protein expression lysate. This mixture was incubated on a rotator at 4 °C for 2 hours prior to addition of glutathione sepharose and incubation for a further hour. Sepharose beads were collected by centrifugation (300 × g), washed 3 times in cold lysis buffer and prepared for SDS PAGE by the addition of sample buffer. For GST pull-downs of recombinant 6His-tagged proteins, normalised amounts of GST- and 6His-tagged protein expression lysate was added to 500μl cold blocking solution (lysis buffer containing 2% w/v skimmed milk, clarified by centrifugation at 13,000 rpm in a microfuge). These pull-downs were then treated as described for cell lysate GST pull-downs.