Fig. 1

PLZF interacts with SIRT1 and HDAC3. Cellular coimmunoprecipitation of PLZF with deacetylases (a). Endogenous coimmunoprecipitation of PLZF and SIRT1 (a.1). Antibodies to PLZF and SIRT1 were used to precipitate each protein from 2 × 10⁷ KG1a cells as noted, and precipitates immunoblotted for PLZF (top panel) or SIRT1 (bottom panel) independently. Endogenous coimmunoprecipitations of PLZF and HDAC3 (a.2). Whole-cell extracts from KG1 cells were subjected to immunoprecipitation with anti-HDAC3 (HDAC3 IP) and anti-PLZF (PLZF IP) antibodies followed by immunoblotting with monoclonal antibodies raised against HDAC3 antibody (αHDAC3) and PLZF (αPLZF). In vitro mapping of PLZF interaction domains (b). Direct in vitro interaction between PLZF and SIRT1, and PLZF and HDAC3 was mapped by GST affinity chromatography using full length GST-PLZF. b.1 Both SIRT1 and HDAC3 don’t interact with the N-terminus repressor domain of PLZF (GST-BTB/POZ). b.2 Top panel bacterially expressed His-tagged SIRT1 was incubated with bacterially expressed GST, or GST-PLZF, subjected to electrophoretic separation and immunoblotted with an antibody against the HIS epitope tag. GST1–5, PLZF zinc fingers 1–5 only; GST-PLZF, full-length PLZF; GST3–9, zinc fingers 3–9 only; GST1–9, zinc fingers 1–9 only. Input, 5 % of volume of SIRT1 sample used in each pull-down. Lower panel ponceau staining of the blot, indicating amount of each protein loaded. Asterisks indicate the GST-fusion species in each lane. b.3 Direct in vitro interaction was mapped by GST or in vitro immunoprecipitation using GST, GST-PLZF or ³⁵S-labeled PLZF translated using the rabbit reticulocyte system (³⁵S-PLZF) and incubated with ³⁵S-labeled HDAC1 (³⁵S-HDAC1) and HDAC3 (³⁵S-HDAC3). Bottom panel GST pull-down using the GST3–9, zinc fingers 3–9; GST1–9, zinc fingers 1–9; GST-PLZF, full length PLZF and GST only. Anti-PLZF antibody (αPLZF) was used for coimmunoprecipitation to evaluate interactions between the PLZF, HDAC1 and HDAC3 proteins