was carried out with a commercially available assay kit (Cayman Chemical, Ann Arbor, MI) with modification. In brief, sub- confluent cells were washed twice and collected in PBS. Cells were solubilized with 20 mM Tris/HCl buffer (pH 7.5) containing 137 mM NaCl, 2 mM EDTA, 10% glycerol, 1%Triton x-100 and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). Cell lysates from different cell types were incubated on ice for 10 min followed by sonication, and then centrifuged at 16000 g for 15 min at 4uC. Same amount of cell lysates (2 mg) from different cell types were then incubated with anti-HDAC6 antibody pre-coupled beads, in that anti-HDAC6 antibody (Santa Cruz Biotech, SC-5258) was coupled to 20% protein G agarose beads (GE Healthcare Life Sciences) in PBS in the presence of 5 mg/ml bovine serum albumin (Sigma, MO) for 6 hours at 4uC, overnight at 4uC. 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Fan LX, Li X, Magenheimer B, Calvet JP (2012) Inhibition of histone deacetylases targets the transcription regulator Id2 to attenuate cystic epithelial cell proliferation. Kidney Int 81: 76–85. 27. Li X, Magenheimer BS, Xia S, Johnson T, Wallace DP, et al. (2008) A tumor necrosis factor-alpha-mediated pathway promoting autosomal dominant poly- cystic kidney disease. Nat Med 14: 863–868. 28. Shibazaki S, Yu Z, Nishio S, Tian X, Thomson RB, et al. (2008) Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1. Hum Mol Genet 17: 1505–1516. PLOS ONE | www.plosone.org 12 November 2012 | Volume 7 | Issue 11 | e49418