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. Author manuscript; available in PMC: 2009 May 20.
Published in final edited form as: Mol Cell. 2007 Jul 20;27(2):197–213. doi: 10.1016/j.molcel.2007.05.033

Figure 3. HDAC6 Deacetylates Cortactin in vivo.

Figure 3

(A) Left panel, Myc-tagged cortactin was expressed in HeLa cells by transient transfection. Various amounts of Flag-tagged HDAC6 and Flag-tagged HDAC5 were expressed in HeLa cells by adenoviral infection. Right panel, Myc-cortactin, Flag-HDAC6, and Flag-tagged HDAC6 catalytically defective mutant (H216/611A) were expressed in HeLa cells by transfection. Anti-Myc immunoprecipitates were Western blotted with antibodies specific to either acetyl-lysine (top panels) or Myc (middle panels). Cell extracts were Western blotted with a Flag-specific antibody (bottom panels). Relative level of acetylated cortactin: Ac-cortactin/cortactin = 0.83 (lane 1, left panel), 0.17 (lane 5, left panel), 0.8 (lane 6, left panel), 0.9 (lane 10, left panel), 0.65 (lane 1, right panel), 0.03 (lane 4, right panel), 0.57 (lane 7, right panel). (B) Left panel, lysates prepared from A549 cells and A549 cells stably expressing HDAC6 siRNA (HD6KD) were immunoprecipitated with a cortactin antibody, and the immunoprecipitates were Western blotted with an acetyl-lysine-specific antibody (top) or an anti-cortactin antibody (second from the top). Lysates were also directly Western blotted with antibodies specific for HDAC6, α-tubulin, and acetylated α-tubulin. Right panel, cell lysates were prepared from stably transfected HeLa cells expressing either the empty vector or high levels of HDAC6 and used either in a high stringency immunoprecipitation with anti-acetyl-lysine-specific antibodies followed by Western blotting with anti-cortactin (top) or for Western blotting with antibodies specific for either cortactin or HDAC6 (middle and bottom panels, respectively). (C) Four different sets of SIRT2 siRNA from the siGENOME SMART pool were purchased from Dharmacon and used to knock-down SIRT2 expression in A549 or HDAC6KD cells. Protein expression levels and the effects on cortactin acetylation were examined by Western blotting with the indicated antibodies. * indicates a non-specific band on the anti-SIRT2 Western blot. (D) Cell lysates prepared from three different ovarian cancer cell lines subjected to Western blot analysis with the indicated antibodies.