Figure 1

FilGAP interacts with mTORC1/2. (A) HEK293T cells were transfected with Flag-FilGAP and Myc-Raptor. After precipitation with Flag antibody, coprecipitated Raptor was detected with anti-Raptor antibody. (B) HEK293T cells were transfected with Flag-FilGAP (WT or V734Y) and Myc-Rictor. After precipitation with HA antibody, coprecipitated Rictor was detected with anti-Rictor antibody. (C) The purified recombinant GST-FilGAP (373-748aa) or GST alone were coupled to glutathione sepharose, and incubated with HEK293T cells lysed in buffer containing CHAPS. The washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (D) GST-FilGAP (373-748aa) coupled to glutathione sepharose were incubated with HEK293T cells lysed in buffers containing CHAPS or Triton X-100. The washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (E) Relative amount of coprecipitated Raptor, mTOR and Rictor in (E) was shown. (F) siRNAs against Raptor or Rictor were transfected HEK293T cells. 72 h later, cells were lysed in buffer containing CHAPS. Cell lysates were incubated with GST-FilGAP coupled to glutathione sepharose, and the washed precipitates were immunoblotted for the presence of Raptor, Rictor and mTOR. (G) HEK293T cells were treated with 100 nM Rapamycin or 500 nM Torin-1 for 1 h. Cell lysates were subjected to GST pull down assay with GST-FilGAP 373-748aa. (H) The amount of Raptor, Rictor, and mTOR precipitated with FilGAP in (G) was calculated and presented as the mean ± SE. *P < 0.05 **P < 0.01 (Student’s t-test). (I) HEK293T cells were cultured in EBSS without amino acids and serum for 2 h, and then incubated in DMEM medium or DMEM medium with insulin for 30 min. Cell lysates were subjected to GST pull down assay with GST-FilGAP 373-748aa. (J) The amount of Raptor, Rictor, and mTOR precipitated with FilGAP in (I) was calculated.