Fig. 4

TCG directly bound Rap1 in BMMs. A Depiction of the chemical structure and chemical synthesis process of TCG-biotin. B BMMs were induced to osteoclasts while simultaneously treated with indicated doses of biotin, TCG or TCG-biotin separately. Osteoclasts were visualized using TRAP staining (Scale bar = 500 µm). C Schematic of the small molecule pull-down assay. Cell lysates of BMMs, with or without pre-treatment of TCG, were subjected to pull-down assay using biotin beads or TCG-biotin beads, followed by mass spectrometry analysis. D Representative mass spectrum of RAP1b. E Validation of the binding between TCG-biotin and RAP1 through small molecule pull-down assay and western blot. F Binding model of TCG-Rap1b. In panorama, TCG's atoms appeared as balls and rods, with carbon atoms in green, hydrogen atoms in white, and oxygen atoms in red. The entire protein was shown as a cartoon, while the amino acids at the binding site were represented as lines. In the enlarged view, ionic bonds are highlighted, with purple lines denoting hydrogen bonds, green lines indicating π–π stacking, and red lines representing π-cation interactions. G BMMs were stimulated with RANKL and M-CSF, and simultaneously co-treated with 1 µM 8-pCPT-2′-O-Me-cAMP-AM and 1 µM TCG for 48 h, transcript levels of genes involved in osteoclast differentiation were assessed by RT-PCR. Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.01