Fig. 3

Runx2 recruited p300 and NCOA3 to assemble a complex in vivo and in vitro

(A) NCOA3 pulled down both Runx2 and p300 in vivo. Equal weights (0.05 g) of three independent IVDs from LPS-challenged mice were mixed to make a homogenate, followed by immunoprecipitation with anti-NCOA3- and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-NCOA3, anti-Runx2, and anti-p300 antibodies. (B) p300 pulled down both Runx2 and NCOA3 in vivo. The same homogenate used in (A) was immunoprecipitated with anti-p300- and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-P300, anti-Runx2, and anti-NCOA3 antibodies. (C and D) In vitro Co-IP results. Different combinations of Myc-tagged and Flag-tagged plasmids, as shown in the figure, were co-transfected into NP-1 cells. After incubation at 37 °C for 48 h, cells were used for immunoprecipitation using anti-Flag-agarose. The input and output proteins were detected with anti-Flag and anti-Myc antibodies. (C) Determination of the direct interaction of ^FlagRunx2-^Mycp300 and ^FlagRunx2-^MycNCOA3. (D) Determination of the direct interaction of ^Flagp300-^MycRunx2 and ^Flagp300-^MycNCOA3.