Cellular distribution of NF-κB/Rel proteins in peripheral blood neutrophils and monocytes. (A) Unstimulated neutrophils or monocyte-enriched mononuclear cell suspensions from the same donor were submitted to NP40 lysis, and the resulting nuclear-containing and nonnuclear fractions were processed for electrophoresis on 15% Kornberg gels (using 10⁶ cell-equivalents per well, unless otherwise stated) and immunoblotting, as described in Materials and Methods. For p105 and p50, the immunoblot was performed using the no. 1157 anti-p50 antiserum; to allow a better visualization of the p105 band in neutrophil nonnuclear fractions, a longer exposure of the same film is shown in the top panel. For p65/RelA, the immunoblot was performed using an antiserum to the N-terminal region (no. 1207). For c-Rel, 6 × 10⁵ cell-equivalents per well were loaded on the gel and immunoblots were performed using the no. 265 antiserum. As a positive migration control for NF-κB/Rel proteins, a whole-cell sample from unstimulated Jurkat T cells was loaded on the outermost left track (“J”). This experiment is representative of at least three. (B) Immunoblots were performed on the same samples as the ones depicted in (A). Upper panel, 10⁶ cell-equivalents were loaded on a 15% Kornberg gel and the membrane was immunoblotted using an anti-LTA₄ hydrolase antiserum (“LTAH”). Lower panel, 5 × 10⁵ cell-equivalents were loaded on an 18% Kornberg gel and the membrane was immunoblotted using an anti-FLAP antiserum. This experiment is representative of at least three. N, nuclear fractions; NN, nonnuclear fractions; pmn, polymorphonuclear neutrophils; mono, autologous monocyte-enriched suspensions; MW, molecular weight markers (in kD).