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Figure 4 CRP Amplified the Effect of LPS on NF- B Activation (Protocol B)
Quantification of the nuclear expression of the activated NF-B p65 subunit was performed by ELISA and expressed as OD at 450 nm. The experimental conditions are as described in Figure 3. Purified CRP, used for the ELISA at the increasing doses of 5 to 10 to 25 µg/ml, induced a slight but significant increase in NF-B p65 expression (p = 0.002) up to a mean ± SD value of 0.174 ± 0.164 OD with the dose of 25 µg/ml, 75% over the basal value (0.099 ± 0.081 OD). Pretreatment with the NF-B inhibitor PDTC prevented the NF-B activation induced by purified CRP, as well as the use of denatured CRP (25den) (A); *p < 0.05, purified CRP 25 µg/ml versus the basal value; **p < 0.01, purified CRP 25 µg/ml versus PDTC inhibition and denatured CRP (25den). LPS alone, at the low dose of 10 ng/ml, induced a mean ± SD increase in NF-B p65 expression of 0.133 ± 0.126 OD (35% over the basal value;  p = 0.042). Coincubation of purified CRP with LPS induced a further increase in NF-B expression, particularly evident with 25 µg/ml of CRP, up to a mean ± SD value of 0.257 ± 0.162 OD (159% over the basal value), which was significantly greater than that observed with LPS alone ( p = 0.001) or CRP alone ( p = 0.003) (B). Abbreviations as in Figures 1 and 2.
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