Coimmunoprecipitation assay

EL4 or Jurkat T cells were collected and lysed in ice-cold NP40 lysis buffer (Beyotime, Shanghai) for 20 min and centrifuged at 10,000 g for 10 min at 4 °C. For HSP90, raptor and mTOR coimmunoprecipitation experiments, the supernatant fractions were incubated overnight at 4 °C with 2 μg of rabbit anti-HSP90, anti-raptor and anti-mTOR antibody, respectively, and precipitated with protein A/G agarose beads for additional 4 h at 4 °C. The beads were washed with NP40 lysis buffer for four times. The immunoprecipitated proteins were separated by SDS-PAGE, and Western blot was performed with the indicated antibodies.