TLR2-Siglec15 Interaction Signaling

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.