Impaired translocation and reduced GTP activation of Rab27a in CF neutrophils. (A) HC and ΔF508 homozygous CF (CFΔ) neutrophil whole cell lysates, secondary and tertiary granules, or isolated plasma membranes were analyzed by immunoblotting for protein expression of Rab27a, VAMP2, Munc13-4, SNAP23, and STX4. Glyceraldehyde-3-phosphate dehydrogenase and Na⁺/K⁺-ATPase were used as a loading control for whole cell lysates and plasma membranes, respectively. Representative blot images are shown (n = 3 subjects per group). (B) HC or ΔF508 homozygous CF (CFΔ) neutrophils were stimulated with TNF-α (10 ng/mL), and membrane proteins were assayed for Rab27a by immunoblotting. Quantification of immunoblots by densitometry revealed decreased levels of Rab27a on membranes of CF cells (P = .05, paired Student t test, n = 3 subjects per group). (C) To assess the activation levels of Rab27a, cell lysates from TNF-α-stimulated cells were immunoprecipitated with polyclonal rabbit anti-Rab27a antibodies. GTP was quantified using a coupled enzymatic assay and normalized to precipitated Rab27a levels and the 0 time point value (P = .004, P = .03, and P = .003 at 0.5, 1, and 4 mintutes, respectively, Student t test, n = 5 subjects per group). Each measurement in B and C are mean ± SEM from biological replicates.