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Fig. 2 | BMC Immunology

Fig. 2

From: Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signaling

Fig. 2

Analysis of RANK expression in LPS- and RANKL-mediated osteoclastogenesis. a Equal amounts of membrane lysate proteins were used for immunoblotting analyses with antibodies against RANK (~ 90 kDa) and GAPDH (loading control; ~ 37 kDa). Protein levels were quantified by densitometry, corrected for the sample load based on the GAPDH level, and expressed as a fold increase relative to the control lane (−). The results represent one of three experiments performed. b and c Immunostaining with an antibody against RANK was performed in non-permeabilized RANKL (b) and LPS (c) -stimulated osteoclasts. White arrows indicate mature osteoclast. Red arrows indicate mononuclear cells. The results represent one of three experiments performed. d Identification of the time-dependent effect of OPG on LPS-induced osteoclast differentiation. The diagrammatic sketch demonstrates the treatment strategy of RAW cells with LPS (5 μg/mL) and OPG (120 ng/mL). e Representative images of TRAP stained osteoclasts in response to the treatment strategy is shown in panel D. TRAP stained osteoclasts in panels A and C were imaged with a 4× objective (magnification: 40X), and panels B and D were imaged with a 10× objective (magnification: 100X). f The number of TRAP-positive multinucleated osteoclasts were counted in both groups from three different experiments. Statistical analysis was performed to compare the number of osteoclasts in the LPS + OPG group with the control group (LPS). T-test was applied, and the difference between groups is not statistically significant. Scanned uncropped autoradiograms are presented in Additional file 5, Figure S5. Corresponding immunoblots are shown in panel A

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