Figure 5.

FKBP65 is a resident protein of the ER. Subconfluent cultures of FBCs were treated for 3 h with either (A) 10 μg/ml brefeldin A + 10 μg/ml ALLN to “hold” the tropoelastin in the fused ER/Golgi compartment (ALLN is needed to prevent tropoelastin degradation), or (B, C) 10 μM monensin to “hold” the tropoelastin in the cis-Golgi cisternae. Cells were then fixed, permeabilized, and double labeled with a monoclonal antibody to tropoelastin (red) and a polyclonal antibody to either (A, B) FKBP65 (green) or (C) Grp94 (green). After treatment with BFA+ALLN, tropoelastin and FKBP65 were colocalized in the fused ER/Golgi apparatus. When tropoelastin was accumulated in the Golgi, however, the two proteins showed distinct patterns of immunolabeling with tropoelastin in the peri-nuclear Golgi region and FKBP65 remaining in the ER. The same distribution of labeling was seen when Grp94 was colocalized with tropoelastin after monensin treatment. Note that the production of tropoelastin by FBCs in culture is density-dependent, thus not all the cells are synthesizing tropoelastin at subconfluency.