Fig. 3.

Determinants of secretion and GPI anchoring of prostasin in mouse lung epithelial cells. MLE-12 cells were transiently transfected with mutated prostasins, separated by SDS-PAGE, and immunoblotted for prostasin. A: conditioned medium was concentrated and normalized to load equivalent volumes; results were identical when loading was normalized to cell number. B: proteins were extracted from MLE-12 cells by Triton X-114 detergent phase separation and precipitated with 15% TCA for analysis. Samples were normalized to cell number for immunoblotting. C: MLE-12 cells were treated with PI-PLC to release cell surface GPI-anchored proteins. Conditioned medium was concentrated, and gel loading was normalized to cell number. All experiments were done at least 3 times, and representative blots are shown. Mock transfected MLE-12 cells (no plasmid) do not express prostasin.