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Fig. 2. LAP2ß binds HDAC3 and both proteins are co-localized to the nuclear membrane. (A) Diagrams representing the LAP2ß and LAP2
protein structures. Numbers represent the corresponding amino acids. The LAP2ß-specific region (amino acids 219-328) that served as bait in the two-hybrid screen is delineated in bold stripes. (B) Schematic representation of the two-hybrid positive clones: 4.4 (0.6 kb) and 4.2 (0.8 kb), and the corresponding full-length cDNAs of HDAC3 and alternatively spliced HDAC3 (GenBank database accession numbers AF074881 and AF074882, respectively). The numbers above the two isoforms represent the corresponding amino acids and the numbers in italics below represent the corresponding nucleotides. The gray-striped area (amino acids 33-70 in both HDAC3 isoforms) represents the sequence common to both positive clones. (C) HDAC3 and LAP2ß bind in vitro. HDAC3 and HDAC1 (as a control) were expressed as GST fusion proteins in bacteria. LAP2ß was translated in vitro in the presence of [35S]-labeled methionine (10% of input, lane 1). Mixtures of [35S]-labeled LAP2ß with GST, GST-HDAC3 or GST-HDAC1 (lanes 1, 2 and 3, respectively) were incubated with glutathione-Sepharose beads. The bound proteins were eluted, separated by SDS-PAGE and identified by autoradiography (top) and Coomassie-Blue staining (bottom). (D) LAP2 does not bind HDAC3. HDAC3 was expressed as a GST fusion protein in bacteria. LAP2 was translated in vitro in the presence of [35S]-labeled methionine (lane 1). Mixtures of [35S]-labeled LAP2 with GST or GST-HDAC3 (lanes 1 and 2, respectively) were incubated with glutathione-Sepharose beads. The proteins were eluted, separated by SDS-PAGE and identified by autoradiography (top) and Coomassie-Blue staining (bottom). (E) Overexpressed HDAC3 and LAP2ß bind in vivo. U2OS cells were co-transfected with HDAC3 (1 µg) and LAP2ß (1 µg). Cell extracts were immunoprecipitated with anti-LAP2ß antibodies (lanes 3 and 6) followed by immunoblotting with anti-HDAC3 (lanes 1-3) or anti-LAP2ß (lanes 4-6) antibodies. Beads only were used as a control for non-specific binding (lanes 2 and 5). A whole-cell lysate was used as a control marker for HDAC3 and LAP2ß (lanes 1 and 4, respectively). (F) Endogenous binding of LAP2ß and HDAC3 upon PHA stimulation of PBMCs. 2x106 PBMCs were stimulated with 25 µg PHA. After 72 hours, cellular extracts were immunoprecipitated with anti-LAP2ß antibodies (lanes 3 and 6), followed by immunoblotting with anti-HDAC3 (lanes 1-3) or anti-LAP2ß (lanes 4-6) antibodies. Beads only were used as a control for non-specific binding (lanes 2 and 5). A whole-cell lysate was used as a control marker for HDAC3 and LAP2ß (lanes 1 and 4, respectively). (G) Co-localization of transfected HDAC3 and LAP2ß. U2OS cells were transfected with either HDAC3 alone (1.0 µg, i), HDAC3 and increasing doses of LAP2ß (0.1 µg, ii; 0.5 µg, iii; 1.0 µg, iv), HDAC1 alone (1.0 µg, v) or HDAC1 and LAP2ß (1.0 µg, vi). The cells were fixed and immunostained with anti-HDAC3, anti-HDAC1 and anti-LAP2ß antibodies, as indicated, and analysed by confocal microscopy.
