Figure S3. Supplemental data of Fig. 4 .... | Rockefeller University Press

Figure S3.

$Supplemental data ofFig. 4. (A and B) Western blotting of the maturation of prApe1 in cells expressing N-terminally (A) or C-terminally (B) truncated Atg15 mutants. atg15Δ cells expressing WT Atg15 or fusion of truncated Atg15 with CPYN50 under the control of ATG15 promoter from a multicopy-plasmid (pRS426) were cultured in the same conditions as in Fig. 2 D. A series of N- and C-terminally truncated forms of Atg15 were constructed by PCR from pRS426-CPY(1–50)-ATG15(Δ1–35)-mNeonGreen (YPL073) (see Materials and methods). (C) Vacuolar pellet fraction (Fig. 4 C, lane 3) was treated with 2% Tx or 0.5% DDM (T2) and incubated at 4°C for 30 min. Then samples were centrifuged at 135,000 g for 30 min, followed by separation into pellet (P2) and supernatant (S2) fractions before being subjected to western blot analysis with α-FLAG antibodies. *, nonspecific band. (D) Vacuolar pellet fraction (Fig. 4 C, lane 3) was treated with 0.5% DDM or 2% Tx. Samples were then subjected to western blotting with α-FLAG antibodies. *, nonspecific band. (E) Lipase activities of vacuolar pellet fraction treated with detergents. NBD-PE was incubated with each fraction in D for 20 min. Final DDM and Tx concentrations in the reaction mixture were 0.05% and 0.2%, respectively. Source data are available for this figure: SourceData FS3.$

Supplemental data of Fig. 4 . (A and B) Western blotting of the maturation of prApe1 in cells expressing N-terminally (A) or C-terminally (B) truncated Atg15 mutants. atg15Δ cells expressing WT Atg15 or fusion of truncated Atg15 with CPY^N50 under the control of ATG15 promoter from a multicopy-plasmid (pRS426) were cultured in the same conditions as in Fig. 2 D. A series of N- and C-terminally truncated forms of Atg15 were constructed by PCR from pRS426-CPY(1–50)-ATG15(Δ1–35)-mNeonGreen (YPL073) (see Materials and methods). (C) Vacuolar pellet fraction (Fig. 4 C, lane 3) was treated with 2% Tx or 0.5% DDM (T₂) and incubated at 4°C for 30 min. Then samples were centrifuged at 135,000 g for 30 min, followed by separation into pellet (P₂) and supernatant (S₂) fractions before being subjected to western blot analysis with α-FLAG antibodies. *, nonspecific band. (D) Vacuolar pellet fraction (Fig. 4 C, lane 3) was treated with 0.5% DDM or 2% Tx. Samples were then subjected to western blotting with α-FLAG antibodies. *, nonspecific band. (E) Lipase activities of vacuolar pellet fraction treated with detergents. NBD-PE was incubated with each fraction in D for 20 min. Final DDM and Tx concentrations in the reaction mixture were 0.05% and 0.2%, respectively. Source data are available for this figure: SourceData FS3.