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Fig. 4. Subcellular localization of glycine receptor (GlyR)α2 in human neutrophils. (A) Isolated neutrophils were pressurized with N2 gas for 20 min at 350 psi, and cavitated neutrophils were loaded onto three separate Percoll density gradients. After centrifugation, four distinct bands were observed from the bottom, designated as α-band (azurophil granules), β1-band (specific granules), β2-band (tertiary granules), and γ-band (secretory vesicles and plasma membranes). For negative controls, non-transfected HEK 293 cells were used. For positive controls, neutrophils and GlyRα2-transfected HEK 293 cells (HEK+α2) were used. Western blot revealed the localization of GlyRα2 in the γ-band (secretory vesicles and plasma membranes), with or without blocking peptide against GlyRα2. Exposure of neutrophils to E. coli or phorbol myristate acetate (PMA) effectively translocated GlyR to the plasma membrane. Representative fluorescein isothiocyanate (FITC) histograms on membrane expression of GlyRα2 in response to E. coli (B) or PMA (C). Neutrophils were treated with E. coli or PMA for 5 min. After fixation, neutrophils were stained with FITC-conjugated anti-GlyRα2 antibody for 30 min. (D) A representative FACS histogram on membrane expression of flavocytochrome b588 in response to E. coli. Neutrophils were exposed to E. coli for 20 min.
Korean J Physiol Pharmacol 2022;26:229-238
© Korean J Physiol Pharmacol