Figure 4.
Inhibition of S6 kinase Thr 389 phosphorylation by Autophagy-specific gene 1α. (A,B) HEK 293T cells were transfected with HA–Akt (A) or Myc–RSK (B), and/or Flag–ATG1α WT or KI plasmids. The immunoprecipitated Akt and RSK proteins were analysed by immunoblot using appropriate antibodies (top three panels). Flag (ATG1α) blots were completed from the same cell lysates (bottom panel). (C) HEK 293T cells were transfected with HA–S6K Thr 389 Glu mutant and/or Flag–ATG1α WT or KI. The immunoprecipitated S6K mutants were analysed by immunoblot using anti-phosphospecific S6K Thr 229 (top panel) and HA (S6K) antibodies (middle panel). Flag (ATG1α) blot was completed from the same cell lysates (bottom panel). ATG1, Autophagy-specific gene 1; EGF, epidermal growth factor; HA, haemagglutinin; KI, ATG1α Lys 46 Ile (kinase-dead) mutant; RSK, ribosomal S6 kinase; WT, wild type.