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. 2023 Aug 28;42(19):e112814. doi: 10.15252/embj.2022112814

Figure 9. TOR‐mediated Ypt1 phosphorylation regulating autophagy is conserved in mammals.

Figure 9

  • A
    In vitro kinase assays were performed using purified GST‐Rab1 from E. coli as substrates, with purified FLAG‐Raptor from nutrient rich‐cultured HEK293T cells as protein kinase. The phosphorylation level of GST‐Rab1A was detected using an anti‐thioP antibody.
  • B
    In vitro kinase assays were performed using purified GST‐Rab1 or Rab1A T75A from E. coli as substrates, with purified FLAG‐Raptor from nutrient rich‐cultured HEK293T cells as protein kinase. The phosphorylation level of GST‐Rab1A WT and T75A variants were detected using an anti‐thioP antibody.
  • C
    The expression levels of Rab1 in NC (non‐specific control) and Rab1 stable knockdown HEK293T cells were analyzed using western‐blot with an anti‐Rab1A or anti‐Rab1B antibodies, respectively. The expression levels of p62 were detected using anti‐p62 antibody. β‐actin served as a loading control.
  • D–G
    (D, F) Rab1 KD stable HEK293T cell lines were transfected with the retroviral vectors encoding FLAG‐Rab1A WT, FLAG‐Rab1A T75D , or with the corresponding empty retrovirus (mock). These cells were cultured in complete medium with 500 nM Torin treatment (D), or 250 nM Torin and 100 nM bafilomycin A1 treatment (F) for 120 min. Cell lysates were then analyzed by western‐blot with anti‐p62 and LC3 antibodies. β‐actin served as a loading control. (E, G) Autophagy activity from (D, F) were quantified by the ratio of LC3II/Actin and p62/Actin, and presented as mean ± SD (n = 3). ***P < 0.001; **P < 0.01; *P < 0.05; NS, no significance; two‐tailed Student's t‐tests were used.
  • H
    Rab1 KD stable HEK293T cell lines were co‐transfected with HA‐FIP200, FLAG‐ULK1, and the retroviral vectors encoding FLAG‐Rab1A WT, FLAG‐Rab1A T75D , or with the corresponding empty retrovirus (mock). These cells were cultured in complete medium with 0.25 mM Torin treatment for 0 or 2 h. Cell lyissates were immunoprecipitated with anti‐HA agarose beads and then analyzed by western blot using an anti‐FLAG antibody.
  • I
    Rab1 KD stable HEK293T cell lines were co‐transfected with HA‐ULK1, and the retroviral vectors encoding FLAG‐Rab1A WT, FLAG‐Rab1A T75D , or with the corresponding empty retrovirus (mock). These cells were cultured in nutrient‐rich medium with mTOR inhibitor Torin treatment for 0 or 2 h. Cell lysates were immunoprecipitated with anti‐FLAG agarose beads and then analyzed by western blot using an anti‐HA antibody.

Source data are available online for this figure.