Fig. 6
USP24 interacted with pRunx2S28 and protected pRunx2S28from proteasomal degradation
(A) Protein levels of USP24 in LPS-treated IVDs and LPS-treated NP-1/AF-1 cells. The protein samples used to detect the USP24 protein levels were the same ones used in Fig. 4A C. (B) USP24 pulled down pRunx2S28 but not Runx2 in LPS-IVDs. Equal weights (0.05 g) of three independent IVDs from LPS-challenged mice were mixed to make a homogenate, followed by immunoprecipitation with anti-USP24-coated and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-USP24, anti-pRunx2S28, and anti-Runx2 antibodies. (C and D) USP24 pulled down pRunx2S28, but not Runx2, in LPS-treated NP-1/AF-1 cells. Cell lysates from LPS-treated NP-1/AF1 cells were used for immunoprecipitation with anti-USP24- and IgG-coated protein A agarose. The purified complexes were used for western blotting assays with anti-USP24, anti-pRunx2S28, and anti-Runx2 antibodies. (E) In vitro Co-IP results. The MycUSP24 plasmid was cotransfected with FlagRunx2 or FlagpRunx2S28D into NP-1 cells. After incubation at 37 °C for 48 h, the cells were used for immunoprecipitation using anti-Myc-agarose. The input and output proteins were detected with anti-Flag and anti-Myc antibodies. (F) MG132 blocked the degradation of pRunx2, which is dependent on USP24 depletion. The Control-KD1/2 and USP24-KD1/2 cells were co-treated with LPS and MG132 for 6 h, followed by protein isolation and western blotting to determine the protein levels of USP24, Runx2/pRunx2, pRunx2S28, and GAPDH