Mab2250

The in vitro protein interactions using recombinant proteins were performed as follows. GST-tagged ANKRD1 (100 ng), HIS-tagged JUN (truncated form 1-241aa, 100 ng), full-length JUN (100 ng), full-length FOS (100 ng), AP-1 DNA oligo (100 ng) (sc-2501, Santa Cruz), or T-5224 (20uM) (MedChemExpress) were mixed with Glutathione-conjugated beads in binding buffer (150 mM NaCl, 100 mM Tris ph8, 0.5% NP40, 10% Glycerol) for overnight (ON) at 4 °C on a rotating platform. After this time the complexes were washed 3 times in binding buffer, then resuspended in 30 µl of SDS-PAGE loading buffer for electrophoresis and heated at 95 °C for 10 minutes. We carried out the western blots by using anti -ANKRD1, -JUN, and -FOS antibodies (reported above) To determine the requirement of a native configuration the recombinant JUN was heated for 20 min in a thermal block at 98 °C, while the same amount of ANKRD1 or FOS proteins were left on ice as control, before mixing them to the pre-heated/denatured JUN protein. Western blot was carried-out using anti-ANKRD1 (sc-365056; Santa Cruz), anti-JUN (mAb #9165; Cell Signaling), and anti-FOS (mAb #2250, Cell Signaling). The concentration/dilution of each antibody is reported in Supplementary Data File 8.

Primary BMMs were isolated for CHIP assay according to the instruction from the Pierce Agarose CHIP Kit (Cat. 26156, Thermo Fisher) using a CHIP-grade antibody to c-Fos (mAb2250, Cell Signaling). In brief, we crosslinked the cell pellet using Glycine Solution after fixation in 1% formaldehyde. The cells were lysed in membrane extraction lysis buffer and nuclear extraction lysis buffer, along with MNase digestion (DTT, MNase Digestion Buffer). Of the sample, 10% was removed as an input control. CHIP was performed according to the protocol provided by manufacturers using an antibody to c-Fos. Anti-RNA polymerase II and control IgG were used as positive and negative controls, respectively. The purified DNA was then analyzed by PCR and electrophoresis.

The knee joint tissues of the mice were collected after upon killing and fixed in 10% buffered formalin phosphate for 48 hr. Samples were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 2 weeks and embedded in paraffin or optimal cutting temperature (OCT) compound (Sakura Finetek). Four μm thick, sagittal-oriented sections of paraffin-embedded knee joints were processed for Tartrate-resistant acid phosphatase (TRAP) staining using a standard protocol (Sigma-Aldrich). Ten μm thick, sagittal-oriented sections of OCT-embedded knee joints were processed for immunofluorescence staining. The sections were incubated with primary antibodies against RANK (1:100, ab13918, Abcam), TLR2 (1:100, mAb12276, Cell Signaling), Maackia Amurensis Lectin (1:100, B-1265–1, Vector Laboratories), ST3GAL4 (1:100, 13546–1-AP, Proteintech), c-Fos (1:100, mAb2250, Cell Signaling), or ST3GAL1 (1:50, PA5-21721, Thermo Fisher Scientific) overnight at 4 °C. Then, the samples were incubated with secondary antibodies and DAPI (1:250, H-1200, Vector) in the dark for 1 hr at room temperature. The fluorescence images were taken by fluorescence microscopy (Olympus BX51, DP71) or confocal microscopy (Zeiss LSM 880) and analyzed by ImageJ software (National Institutes of Health, Bethesda).