Fig. 2

SIRT1 and HDAC3 deacetylase PLZF protein. Detection of PLZF by Western blotting (a). PLZF is deacetylated by SIRT1 (a.1). Expression constructs were transfected as indicated into 293T cells, whole cell lysates were extracted and immunoprecipitated with an antibody to the FLAG-tagged PLZF construct. Precipitates were immunoblotted with an antibody to acetylated PLZF species (top panel α-AcK) or FLAG (bottom panel α-FLAG). HDAC3 deacetylates PLZF (a.2). Expression constructs were transfected as indicated into 293T cells, whole cell lysates were extracted and immunoprecipitated with an antibody to the FLAG-tagged PLZF construct. Precipitates were immunoblotted with an antibody to acetylated PLZF species (top panel α-AcK) or FLAG (bottom panel α-FLAG). Detection of PLZF signatures by nanoimmunoassay. Electropherograms depicting levels of total and acetylated PLZF protein (b). Beta-2 microglobulin was used as loading control. Monoclonal PLZF antibody detects both acetylated (grey arrows) and non-acetylated (green arrow) forms as treatment by a p300 inhibitor (anacardic acid) induces the reduction of pics marked by grey arrows. Histogram plot (c) showing the ratio of acetylated versus non-acetylated forms of PLZF under the different conditions (PLZF only: PLZF; anacardic acid treatment: PLZF + p300i; co-expression of PLZF and HDAC3: PLZF + HDAC3 and co-expression of PLZF and SIRT1: PLZF + SIRT1). Each reaction was done in triplicate