Figure 6. HDAC1 deacetylates p150^Glued at lysine 230.

A. MS/MS spectrum of acetylated p150^Glued tryptic peptide spanning lysine 230. Annotated product ions highlighted in red and blue correspond to b- and y- type ions, respectively. Neutral loss ions from the peptide precursor or product ions are highlighted in green. The observed product ions confirmed acetylation at K230 as indicated by a 42 Da mass shift.

B. (Top) Schematic of p150^Glued. (Bottom) Comparison of protein sequences from the DIC binding region of CC1 around K230 from the indicated species.

C. Inhibition of HDAC1 increases the acetylation of p150^Glued. (Top) HEK293 cells were treated +/− MS275 for 48 hrs, fractionated and cytosolic lysates were immunoprecipitated with anti-acetyl lysine and western blotted for p150^Glued. (Bottom) Bar graph showing the average ratio of acetylated over total p150^Glued ± SEM for n=7; *, p < 0.05, student’s t-test.

D. Over expression of HDAC1 reduces the amount of acetylated p150^Glued. (Top) HEK293 cells were transfected with Myc-p150^Glued with or without HDAC1-Flag. The cytosolic lysates were immunoprecipitated with anti-acetyl lysine and western blotted with an antibody to Myc. (Bottom) The average ratio of acetylated over total p150^Glued ± SEM for n=5; **, p < 0.01, student’s t-test.

E. Activation of p75NTR reduces the level of acetylated p150^Glued. Sympathetic neurons were treated with 12.5 mM KCl or 200 ng/ml BDNF +/− 5 μm MS275 (MS) for 48 hrs. The neurons were fractionated and the cytosolic lysates were immunoprecipitated with anti-acetyl lysine and western blotted for p150^Glued or alpha-Tubulin. (Top) Representative western blot image. (Bottom) The average ratio of acetylated over total p150^Glued or tubulin ± SEM for n=6–7; *, p < 0.05, student’s t-test.