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. 2010 Sep 20;190(6):1005–1022. doi: 10.1083/jcb.200912089

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© 2010 Mari et al.

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Figure 7. — Subcellular fractionation of Atg9 clusters in wild-type, atg11Δ, and atg1Δ backgrounds. (A) The Atg9-PA (FRY172), Atg9-PA atg11Δ pep4Δ (FRY250), and Atg9-PA atg1Δ pep4Δ (FRY196) strains were grown to log phase, converted to spheroplasts, and lysed, then cell extracts were centrifuged at 13,000 g for 15 min. The cell extract (T), the supernatant (S13), and the pellet (P13) fractions were then separated by SDS-PAGE, and Atg9 distribution was analyzed by Western blotting using anti-PA antibodies. Efficient lysis and correct fractionation were assessed by verifying the partitioning of cytosolic Pgk1 and mitochondrial Por1. M_r is indicated in kilodaltons. (B) The S13 supernatants were fractionated on sucrose step-density gradients, and the fractions were analyzed by Western blotting using antisera against PA (for Atg9-PA), Pep12, Tlg1, and Pgk1. (C) Quantification of the immunoblots.