Figure 2. Cortactin is Acetylated in vivo.

(A) HeLa cells transfected with either an empty vector or a vector encoding Myc-tagged cortactin were treated with ethanol (−) or 400 ng/ml TSA (+). Anti-Myc immunoprecipitates were Western blotted using antibodies specific for acetyl-lysine (AcK) or Myc. Relative level of acetylated cortactin: Ac-cortactin/cortactin = 0.33 (lane 2), 1.75 (lane 3). (B) HeLa cells were untreated (bottom panel) or treated (top panel) with either ethanol (−) or 400 ng/ml TSA (+) for 12 h. Lysates were immunoprecipitated under high stringency conditions and immunoprecipitates were Western blotted with the indicated antibodies. The same lysates were directly immunoblotted with an anti-cortactin antibody. (C) Left top panel, total extracts prepared from HeLa cells treated with ethanol (−) or 400 ng/ml TSA plus 20 mM nicotinamide (+) for 12 h were separated on SDS-PAGE and analyzed by Western blot with the anti-acetyl-cortactin antibody. The blot was stripped and re-probed with an anti-cortactin antibody. Left bottom panel, HeLa cells transfected with plasmids expressing Flag-tagged cortactin or an empty parental vector were treated with ethanol (−) or 400 ng/ml TSA plus 20 mM nicotinamide (+) for 12 h. Cell lysates were immunoprecipitated with anti-Flag and Western blotted with an anti-acetyl-cortactin antibody or directly Western blotted with a Flag-specific antibody. Right panels, extracts prepared from cells treated with nicotinamide, sodium butyrate (NaB), and/or TSA were analyzed as before. Anti-Ac-H4 blot was performed to confirm that NaB was active in this system. Anti-cortactin and anti-β-actin Western blots were done as loading controls. (D) Top panel, anti-cortactin immunoprecipitates prepared from a NIH3T3 whole cell extract was separated on a 2-dimensional gel and Western blotted with an anti-acetyl-lysine antibody. Bottom panel, the blot was stripped and re-probed with an anti-cortactin antibody.