Skip to main content
. 2015 Jul 2;34(17):2272–2290. doi: 10.15252/embj.201590992

Figure 3.

Figure 3

mTORC1 phosphorylates the UVRAG–VPS34 lipid kinase complex

  1. HeLa, U2OS and MEFs were treated in complete medium (control), 1 μM KU0063794 (KU) or EBSS for 1 h (MEFs) or 2 h (HeLa, U2OS) prior to lysis and immunoblot.
  2. Cell lysate from MEFs grown in complete medium was treated in the presence or absence of lambda phosphatase with or without phosphatase inhibitors for 30 min at 30°C.
  3. RICTOR +/+ and −/− MEFs were grown in complete medium (control), 1 μM KU or EBSS for 1 h prior to lysis.
  4. Endogenous RAPTOR (RPTOR) or IgG control was immunoprecipitated from HEK293 cells and utilised for an in vitro kinase assay (Materials and Methods) with substrates GST-p70S6K D236A, GST-UVRAG, GST-ATG14L or FLAG-BECLIN1 in the presence or absence of KU.
  5. Quantitation of relative phosphorylation of GST-p70S6K and GST-UVRAG in the presence or absence of KU from (D). Data represents mean ± SEM for n = 3 independent experiments. Significance was determined by Student’s t-test, ***P < 0.001.

Source data are available online for this figure.