Figure 3

A) Western blot analysis of mucin granule immuno-isolations reveals that CSP, HSP70, and MARCKS are associated with the granule. Mucin granules were isolated as described, and isolated granules separated from other whole cell organelles through differential centrifugation in a 86% Percoll gradient and targeted by incubation with the rabbit-anti-mclca3 antibody. Immuno-isolation blots were probed with anti-CSP, anti-HSP70, and anti-MARCKS in unstimulated (Lane C) and PMA-exposed (Lane B) well-differentiated NHBE cell. Cells were exposed to 100 nM PMA for 15 min. Whole molecule rabbit IgG was the negative control used for the immuno-isolations (Lane A). B) CSP, HSP70, MARCKS, and CLCA are associated in NHBE cells. Immunoprecipitation of MARCKS from whole cell lysates followed by immunoblotting for CSP, HSP70, and hCLCA1, in NHBE cells indicates that these proteins appear associated with each other (Lane C). Whole cell lysates (Lane B) also show the presence of these proteins. Immunoblotting with anti-MARCKS antibody was the positive control. Whole molecule rabbit IgG was the negative control used for the immuno-isolations (Lane A).