Figure S2. Supplemental data of Fig. 2 .... | Rockefeller University Press

Figure S2.

$Supplemental data ofFig. 2. (A) Western blotting of vacuolar fractions from WT, atg15Δ, and atg15Δ cells expressing C-terminally FLAG-tagged Atg15 (Atg15-FLAG) from either a single plasmid (pRS316) or a multicopy plasmid (pRS426). The ratio of the band intensities detected by α-FLAG (red squares) and α-Pho8 antibodies for each strain are shown in the right panels. *, nonspecific band. (B) Maturation of prApe1 in cells expressing Atg15 tagged with FLAG in C-terminus. Cells were grown in SD/CA medium and treated with rapamycin for 2 h. prApe1 and mApe1 were detected with α-Ape1 antibodies. Western blotting (left panel) shows representative results from three independent experiments. The band intensities of prApe1 and mApe1 were measured, and the percentage of mApe1 to the sum of prApe1 and mApe1 were determined (right panel). Error bars represent means ± SD of three independent experiments. (C) Lipase activity of vacuolar lysates from the strains in A. Each lysate was incubated with NBD-PE for 20 min. (D) Western blotting of vacuolar fractions from atg15Δ and pep4Δ prb1Δ cells each expressing Atg15-FLAG from a multicopy plasmid. The experimental procedure was the same as in Fig. S1 B. (E) Maturation of prApe1 in mNG-tagged Atg15 expressing cells. Culture condition was the same as in B. prApe1 and mApe1 were detected with α-Ape1 antibodies. Full length of C-terminally mNG-tagged Atg15 and CPYN50-Atg15ΔN35 were detected with α-mNG antibodies. (F) Fluorescence microscopy image of mCherry-Atg8 in WT cells. Cells were grown in SD/CA medium and then treated with rapamycin for 3 h. Scale bar, 1 µm. Dashed lines, vacuole boundaries. DIC, differential interference contrast. (G) Western blotting of vacuolar fractions from atg15Δ and pep4Δ prb1Δ cells each expressing CPYN50-Atg15∆N35. The experimental procedures were the same as in Fig. S1 B. Source data are available for this figure: SourceData FS2.$

Supplemental data of Fig. 2 . (A) Western blotting of vacuolar fractions from WT, atg15Δ, and atg15Δ cells expressing C-terminally FLAG-tagged Atg15 (Atg15-FLAG) from either a single plasmid (pRS316) or a multicopy plasmid (pRS426). The ratio of the band intensities detected by α-FLAG (red squares) and α-Pho8 antibodies for each strain are shown in the right panels. *, nonspecific band. (B) Maturation of prApe1 in cells expressing Atg15 tagged with FLAG in C-terminus. Cells were grown in SD/CA medium and treated with rapamycin for 2 h. prApe1 and mApe1 were detected with α-Ape1 antibodies. Western blotting (left panel) shows representative results from three independent experiments. The band intensities of prApe1 and mApe1 were measured, and the percentage of mApe1 to the sum of prApe1 and mApe1 were determined (right panel). Error bars represent means ± SD of three independent experiments. (C) Lipase activity of vacuolar lysates from the strains in A. Each lysate was incubated with NBD-PE for 20 min. (D) Western blotting of vacuolar fractions from atg15Δ and pep4Δ prb1Δ cells each expressing Atg15-FLAG from a multicopy plasmid. The experimental procedure was the same as in Fig. S1 B. (E) Maturation of prApe1 in mNG-tagged Atg15 expressing cells. Culture condition was the same as in B. prApe1 and mApe1 were detected with α-Ape1 antibodies. Full length of C-terminally mNG-tagged Atg15 and CPY^N50-Atg15^ΔN35 were detected with α-mNG antibodies. (F) Fluorescence microscopy image of mCherry-Atg8 in WT cells. Cells were grown in SD/CA medium and then treated with rapamycin for 3 h. Scale bar, 1 µm. Dashed lines, vacuole boundaries. DIC, differential interference contrast. (G) Western blotting of vacuolar fractions from atg15Δ and pep4Δ prb1Δ cells each expressing CPY^N50-Atg15^∆N35. The experimental procedures were the same as in Fig. S1 B. Source data are available for this figure: SourceData FS2.