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Fig 3 | Molecular Cancer

Fig 3

From: MDM4 actively restrains cytoplasmic mTORC1 by sensing nutrient availability

Fig 3

MDM4 binds and inhibits mTOR kinase activity. a Wb analysis of indicated proteins used for in vitro kinase assay (left panel). Flag-mTOR and GST-S6K were overexpressed an purified from HeLa cells. Right panel shows the levels of MDM4 in the cell input used for the in vitro kinase assay. Histogram in the lower panel shows the ratio of densitometric values of pS6K1 to S6K1. The ratio of pS6K1/S6K1 from siCTL lane was arbitrarily set to 1. Mean ± SD of two independent biological replicates is shown. b Wb analysis of the indicated proteins from in vitro kinase assay. Immunoprecipitated Flag-mTOR and GST-p70S6K1 (GST-S6K) were incubated with GST-MDM4 (purified from bacteria) for 10’ before the kinase assay. Histogram reports data as in (a) (N = 4) c Wb of indicated protein in co-immunocomplexes from HeLa cells transfected with the indicated plasmids. 500 μg of whole cell extract (WCE) was immunoprecipitated with anti-MDM4 antibody C82 (IPαMDM4, left panel). Right panel shows analysis of 1/10 of WCE. d Wb of indicated protein in co-immunocomplexes from HeLa cells transfected as in (c) and after 24 h grown in EBSS (−) for 1 h and then in absence or presence of the aa for additional 15’. e Wb of indicated protein in co-immunocomplexes from HeLa cells grown in EBSS for 50’ (−) and then in absence or presence of the aa for additional 15’. 1 mg of WCE was immunoprecipitated with anti-MDM4 antibody C82 (IPαMDM4) or Ig control (IPCTL). Right panel shows analysis of 1/15 of WCE. f Scheme of MDM4 deletion mutants. P53-BD means p53-binding domain, RF Ring Finger domain. g Wb of indicated protein in co-immunocomplexes from 293 T cells transfected with the indicated plasmids. 500 μg of WCE were immunoprecipitated with anti-mTOR antibody (IPαmTOR) or control Ig (IPCTL). Right panel shows the analysis of 1/10 of WCE. h Wb analysis of the indicated proteins in 293 T cells transfected with the indicated plasmids. After 48 h cells were grown in the medium without aa for 3 h and then in absence (w/o aa) or presence of aa for the last 15’. Histogram as in (a) (N = 2)

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