NFPRA

AcronymDefinition
NFPRANational Forest Protection and Restoration Act
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A 25 kDa fragment is also observed in the upper left blot depicting the bands recognized by NFPRa. However, as expected, it is not sensitive to fMLF and has the same density of stain irrespective of fMLF treatment of the PMN.
The DFP-dependent elimination of the 25K-FPR1 and 25K-FPR1*, recognized by both NFPRb and NFPRa, respectively, supports the hypothesis that this protein band is a C-terminal fragment of FPR1.
NFPRa binding, not being sensitive to FPR1 phosphorylation, was unaffected by NaF treatment (see the boxed area in the upper blot), suggesting that the 25K-FPR and 25K-FPR1* bands were still present irrespective of ATP depletion and their generation was not an energy-dependent process.
Neither of these parameters had an effect on the level of detection of 25K-FPR1 by NFPRa and NFPRb, suggesting the 25K-FPR1 is not produced by a physiologic process of adherent cells.
The right half of the figure shows a blot from samples run on the same gel developed with phosphosensitive NFPRb and the left half shows identical samples developed with the phosphoinsensitive NFPRa. In each gel triplicate, sets of 5 x [10.sup.5] PMN per well were exposed to 1 [micro]M fMLF (+) or vehicle (-) and incubated for 10 min before extraction at room temperature with SDS-containing TS buffer shown on the left half of each blot or at 4[degrees]C with LDS-containing TL buffer shown on the right half of each blot.
To mitigate this variability, the normalized NFPRb density was calculated as the ratio of NFPRb to NFPRa signal for identical samples and is plotted as a percent.
40 [micro]L of the final extract (2.7 x [10.sup.4], 5.3 x [10.sup.4], 8 x [10.sup.4], 10.7 x [10.sup.4], and 13.3 x [10.sup.4] cell equivalents/lane) was then loaded on two 28-well SDS-poly acrylamide gels, immunoblotted, and developed with NFPRa (upper) or NFPRb (lower) as described in Section 2.