TNF-α and fMLP do not activate BLyS intracellular processing. (A) Neutrophils were incubated for 20 hours with or without G-CSF and then were stimulated with TNF-α and fMLP. After 4 hours, cells were lysed, as described in “Patients, materials, and methods.” Whole cell extracts (180 μg) were electrophoresed, blotted, and analyzed for BLyS protein expression using a specific anti-BLyS polyclonal antibody recognizing membrane-bound (arrow at 32 kDa) and soluble BLyS (arrow at 17 kDa) forms. The experiment shown is representative of 2. (B) Neutrophils were cultured for 20 hours with G-CSF and then were stimulated with CXCL8, TNF-α, fMLP, and IC in the absence or the presence of CMK. Culture supernatants were harvested and then processed for BLyS determination by specific ELISA. The experiment depicted is representative of 3. (C) SW neutrophils isolated from patients with RA and healthy subjects were lysed and subjected to immunoblot analysis for intracellular BlyS, as described. The experiment shown is representative of 2.