Sc 21759

For the co-IP assays, cells were first lysed in the presence of protease inhibitors using IP buffer. The lysates were then immunoprecipitated using primary antibodies against mouse TLR2 (Santa Cruz Biotechnology, sc-21759) and Siglec15 (PA5-48221, Thermo Fisher Scientific), followed by absorption on Protein A/G as suggested by the manufacturer (26149, Thermo Fisher Scientific). SDS–PAGE was then used to separate the immunoprecipitates before transfer to a nitrocellulose membrane for immunoblotting procedures. The membranes were incubated with primary antibodies against mouse p-ERK (44-680G, Thermo Fisher Scientific, 1:1 000), ERK (13-6200, Thermo Fisher Scientific, 1:1 000), p-S62-Myc (ab51156, Abcam, 1:1 000), c-Myc (ab32072, Abcam, 1:1 000), TLR2 (Santa Cruz Biotechnology, sc-21759, 1:1 000), and Siglec15 (PA5-48221, Thermo Fisher Scientific, 1:1 000) for 12 h at 4 °C, followed by a 1-h incubation with a secondary antibody (1:1 000). The Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) was used to detect luciferase activity. Enhancer assays were performed with adjustment for transfection efficiency differences.^{59 (link)} Reporter cotransfection assays were performed with a reporter-to-activator plasmid.

Mouse bone specimens were first fixed and then decalcified using 10% EDTA (Sigma-Aldrich, St. Louis, MO, USA) for 14 days with constant shaking. For the histological assays, the detailed protocols were reported in a previous study.⁵⁸ In short, the samples were then dehydrated and embedded in optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) or in paraffin. Four-μm-thick coronal-oriented femur sections were prepared for TRAP staining. Forty-μm-thick coronal-oriented femur sections were prepared for IF staining. The detailed protocols were described in a previous study.⁵⁸ Briefly, the sections were incubated with primary antibodies against mouse TLR2 (Santa Cruz Biotechnology, sc-21759, 1:200), Siglec15 (PA5-48221, Thermo Fisher Scientific, 1:100), ST3GAL1 (PA5-21721, Thermo Fisher Scientific, 1:50), and TRAP (Abcam, ab191406, 1:100) for 12 h at 4 °C. For sialic acid detection, biotinylated Maackia Amurensis Lectin II (MAL II) (Vector Laboratories, CA, USA) was used to label the α2,3 linkage, and biotinylated Sambucus Nigra Lectin (SNA) (Vector Laboratories, CA, USA) was used to label the α2,6 linkage. Fluorescein-conjugated streptavidin (Vector Laboratories, CA, USA) was used for the addition of a fluorescent label to biotinylated sialic acid conjugates. A Zeiss LSM 780 confocal microscope and an Olympus BX51 microscope were used for image capture.