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. Author manuscript; available in PMC: 2014 Jan 8.
Published in final edited form as: Nat Cell Biol. 2013 May 19;15(7):741–750. doi: 10.1038/ncb2757

Fig.3. Beclin-1 is a physiological target of ULK kinase in response to amino acid withdrawal and mTOR inhibition.

Fig.3

Unless otherwise stated all experiments were repeated three times and data shown is representative. (a) Wild-type MEF were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blot was performed with the indicated antibodies. Beclin-1 S14 phosphorylation was quantified (shown under top panel) normalized to total Beclin-1. (b) Wild-type MEF were starved for the indicated time points. Beclin-1 was immunopurified by ATG14L IP and immunoblotted as indicated (top panels). Whole cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed. (c) Wild-type or FIP200 -/- MEF were incubated under nutrient rich, amino acid deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig.3b. (d) Wild-type or ULK def MEF were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in Fig.3a. Two unique experiments were performed.