Mouse 5415

Cells were plated and allowed to attach and grow for 48 h. When cells reached approximately 60% confluency, media was replaced, and cells were allowed to grow for 24–48 h. Media was collected in microcentrifuge tubes and spun down at 12,000 rpm for 3 min at 4 °C to remove debris. The supernatant was transferred to a clean microcentrifuge tube (1 ml/sample), and samples were precleared by adding 10 μl protein A/G beads (Santa Cruz, Cat# SC-2003) for 1 h at 4 °C. Samples were centrifuged at 12,000 rpm for 1 min at 4 °C before transferring the supernatant to a new pre-chilled tube. Then, 1 μl of antibody was added to each sample and rotated for 1 h at 4 °C. For control samples, rabbit/mouse IgG (Cell Signaling, Rabbit #2729 or Mouse #5415) was used as appropriate. In all, 20 μl protein A/G beads were added to the mixture, and the sample was incubated with rotation for 4 h overnight at 4°C. Immunoprecipitated proteins were collected using centrifugation at 12,000 rpm for 1 min at 4 °C. The supernatant was aspirated, and beads were washed in 500 μl of ice-cold lysis buffer three times with centrifugation for 1 min at 12,000 rpm and 4 °C. Samples were eluted from the pellet with 20 μl of loading buffer and heated at 95–100 °C for 5 min. Beads were collected by centrifuging at 12,000 rpm for 1 min. The supernatant was transferred to a gel for electrophoresis.