Supplementary Figure 5: Regulation of the interactions of Atg13 with Atg1 and Atg17.

(a) Western blots showing in vivo Atg1–Atg13 interaction analyzed by coimmunoprecipitation in different buffer conditions. Samples are yeast total lysates (Input) prepared from ATG1–GFP cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +) and immunoprecipitates isolated with anti-GFP magnetic beads (20xEluate) prepared from buffer conditions 1 and 2. Buffer condition 1 is the same as that used in other coimmunoprecipitation experiments performed in this manuscript. Buffer condition 2 is the same as that used in the recent paper by Kraft et al. Asterisks indicate nonspecific bands. Atg13–P indicates phosphorylated forms of Atg13. (b) Western blots using anti-HA antibody showing in vivo Atg1–Atg13 interaction analyzed by coimmunoprecipitation. Samples are yeast total lysates (Input) prepared from ATG1–GFP cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +) and immunoprecipitates isolated with anti-GFP magnetic beads (20xEluate). Atg13–2xHA–P indicates phosphorylated forms of Atg13–2xHA. λPPase indicates treatment of the immunoprecipitates with lambda protein phosphatase. (c) Western blots showing the effects of S429D mutation in Atg13 on the Atg1–Atg13 interaction analyzed by coimmunoprecipitation. Coimmunoprecipitation experiments and western blots were performed as in b. (d) Western blots showing the phosphorylation state of Atg13–2xHA probed with anti-HA antibody and phospho-specific anti-S428/9–P antibodies. Samples are immunoprecipitates of anti-HA immunoprecipitation prepared from cells grown in nutrient-rich medium (Rapamycin, –) or treated with rapamycin for 1 h (Rapamycin, +). (e) Immunoprecipitation and western blots performed as in d. λPPase indicates treatment of the immunoprecipitates with lambda protein phosphatase. Uncropped gel images are shown in Supplementary Fig. 6r–u.