Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases In Vivo
Fig 2
Subcellular localization of proHNP processing.
(A) PLB-985 cells were pelleted and disrupted by nitrogen cavitation. After low speed centrifugation, the cavitate was divided in a postnuclear pellet (P1) consisting of nuclei and unbroken cells and a post-nuclear supernatant (S1) containing cytosol, organelles (including granules), and cell membranes. S1 was underlaid with a two-layer 1.05/1.07 PBS/Percoll density gradient and centrifuged at 37.000g for 30 minutes. Fractions were collected from the bottom of the gradient. Percoll was removed from fractions by ultracentrifugation. (B) Fractions were subjected to Western blotting for HNP, proHNPs, the endoplasmic reticulum (ER) marker calnexin, and the Golgi marker RCAS1. (C) PLB-985 cells were pulsed overnight in medium containing 100 μCi/mL 35S-methionine/cysteine. Cells were pelleted and the supernatant used for isolation of 35S-labelled proHNP by affinity chromatography with an antibody specific for proHNP. Radioactive fractions were pooled, dialyzed against PBS, and tested for proHNP by 16% SDS-Tricine-PAGE and fluorography. (D) 35S-proHNP was incubated with subcellular fractions of PLB-985 for 15 hours at 37°C. Processing was tested by 16% SDS-Tricine-PAGE and fluorography.