Fig. 2From: Lipopolysaccharide- TLR-4 Axis regulates Osteoclastogenesis independent of RANKL/RANK signalingAnalysis 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 ABack to article page