FIGURE 2:

Acetylation of C-terminal lysine residues, K437 and K475, is SIRT1-sensitive. DAOY cells transfected with the following expression plasmids: pcDNA3 vector control, Pax3-pcDNA3, pcDNA3-Pax3ΔK437, pcDNA3-Pax3ΔK475, or pcDNA3-Pax3ΔK437+ΔK475, were cotransfected with SIRT1 cDNA in pcDNA3, pcDNA3 vehicle, SIRT1 siRNA, or control scrambled siRNA. At 48 h posttransfection, total cell lysates were tested for the presence of SIRT1. Lysates were immunoprecipitated (IP) with acetyl-lysine polyclonal antibody and immunoblotted (IB) with Pax3 polyclonal antibody as shown in (A) or immunoprecipitated with Pax3 antibody and immunoblotted with acetyl lysine antibody as shown in (B). Bands were quantified by densitometry and the band intensity (ratio of immunoprecipitated band intensity to input Pax3 band intensity) expressed as arbitrary densitometry units. Results are presented as arbitrary densitometry units + SEM. Each experiment was conducted in quadruplicate and each data point in duplicate.