Figure 2.
AGR2 physically associates with MUC5AC. (A, B) Immature MUC5AC (blue in A) was restricted to AGR2 stained areas (red in B) in tracheal epithelial cells. Purple-stained areas in B represent areas of AGR2/immature MUC5AC colocalization. Gray indicates nuclear (DAPI) staining. Scale bar: 5 μm. (C) Immunoblot probed with antiimmature MUC5AC antibody. Immature MUC5AC was detected in AGR2-containing complexes immunoprecipitated from A549 cell lysates (lane 1). MUC5AC was not detected after immunoprecipitation from A549 cell lysates using nonimmune rabbit serum (lane 2) or after immunoprecipitation of AGR2 from lysates of MG63 fibroblasts, which do not produce MUC5AC (lane 3). Lane 4 contained A549 cell lysate. Samples were subjected to agarose-acrylamide gel electrophoresis under nonreducing conditions, and numbers at the right indicate positions of molecular weight markers (mass in kD). The predicted molecular mass of MUC5AC monomer based on its amino acid composition is approximately 610 kD (41). Covalent modifications, including mannosylation (42), N-glycosylation (6), dimerization, and associations with AGR2 (and possibly other ER proteins), might add to the molecular mass of the immature MUC5AC complex.