Localization and posttranslational modification of fibin analyzed by Western blotting. (A) To analyze the subcellular distribution of fibin cells were harvested 72 h after transfection followed by preparation of the homogenate, the cytosolic and the membrane fraction. 50 μg of protein were applied to each lane. (B) For deglycosylation experiments, fibin expressed in COS-7 cells was incubated either with or without (w/o) PNGase F or not incubated (w/o incubation). Furthermore, the fibin mutant N30Q lacking the N-glycosylation site, was expressed in COS-7 cells and fibin lacking the signal sequence were expressed in E. coli (Fibin E. coli). Immunoblots (A) and (B) were performed with anti-fibin-1-antibodies. (C) Wild-type (WT) fibin, and the fibin mutants C52S, C64S, C52S/C64S were expressed in COS-7 cells. After 72 h, cells were harvested and lysates were prepared. Equal amounts of protein were loaded and separated by SDS-PAGE under either non-reducing or reducing conditions. For immunoblotting the anti-fibin-2 antibody was applied. Negative controls (NC) were prepared as described under Methods.