Figure 2. APPL1 interacts with the NuRD subunits in both cytoplasmic and nuclear fractions independently of HDAC enzymatic activity.

(A) Cytoplasmic (C) and nuclear (N) fractions along with total extracts (T) of three different cell lines, HeLa, HEK-293 and A431, were analysed for the presence of several NuRD subunits by Western blotting with different antibodies as indicated. For detection with a given antibody, equal amounts of proteins from all fractions and three cell lines were loaded (20 μg of protein for blotting with anti-p66α/β, -MBD2/3 and -EEA1 antibodies; 15 μg of protein for anti-APPL1, -HDAC2, -RbAp46 and -GAPDH; 10 μg of protein for anti-MTA2, -HDAC1, -RbAp48 and -Histone H3; the different amounts of protein loaded were chosen to match different sensitivities of the antibodies used). Cytoplasmic (GAPDH and EEA1) and nuclear (histone H3) markers were used to demonstrate the purity of fractions. (B) HeLa cells were transfected for 72 h with two oligonucleotides (a, b) against HDAC2 or with non-specific control oligonucleotide (Φ). Cytoplasmic and nuclear fractions were prepared and subjected to immunoprecipitation (IP) using anti-APPL1 antibody or non-specific immunoglobulins (IgG). Immunoprecipitates were tested for the presence of several NuRD subunits by immunoblotting with various antibodies as indicated. Right panel: 10% of the input material (cytoplasmic and nuclear fractions) were analysed for the knockdown efficiency using anti-HDAC1 and anti-HDAC2 antibodies, as well as for the fraction purity with anti-GAPDH and anti-histone H3 antibodies. (C) Immunoprecipitation from cytoplasmic (C) or nuclear (N) fractions of HeLa cells treated for 20 h with 100 ng/ml of TSA (left panel) or 25 mM sodium butyrate (BUT; right panel) was performed using anti-APPL1, anti-MTA2 or non-specific rabbit IgG. Precipitates along with 10% of the input material were analysed by Western blotting using different antibodies, as indicated. Ctr, control.