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Fig. 5 | BMC Immunology

Fig. 5

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

Fig. 5

Effect of RANKL, LPS, and anti-TNF-α / LPS on the membrane levels of TNFR-1 and TNFR-2. Panels a, c, and d Immunoblotting analysis for TNFR1 (a; top panel, ~ 55 kDa) and TNFR-2 (middle panel in a, ~ 68 kDa) in osteoclasts differentiated with RANKL and LPS are shown. Representative immunoblotting showing the membrane levels of TNFR-1 and -2 (Panel a). Protein levels in untreated RAW cells (−) are shown in lane 1. Membrane levels of TNFR-1 (n = 4) and TNFR-2 (n = 3) were quantified in an Un-Scan IT software, corrected for the GAPDH level, and provided as percentage surface level of receptors (Panels c and d). **p < 0.001 vs. LPS-treated cells (c and d). One-way ANOVA was applied, and values are expressed as mean ± SD of four and three independent experiments for TNFR-1 and TNFR-2, respectively. TNFR-2 blot in A was stripped and blotted with a GAPDH antibody (bottom panel in a). Panels b and e-h Immunoblotting analysis demonstrates the effect of LPS (lane 1) and LPS+ neutralizing antibody to TNF-α (Lane 2) on the membrane levels of TNFR1 (b; top panel) and TNFR-2 (b; middle panel). Protein levels were quantified by densitometry, corrected for the sample load based on GAPDH level, and provided as a fold-change relative to LPS- treated control cells (Panels e and g). The Table (f and h) provides the average pixel value of the protein bands (TNFR-1 in f and TNFR-2 in h) from two experiments and fold changes in the surface levels of interest proteins. The experiment was performed twice and demonstrated remarkably related results. Raw data are provided in the (Additional file 7, Figure S7; A-C)

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