Fig 1. Expression and secretion of TGFBIp in wild-type (WT) and heterozygous (HE) or homozygous (HO) mutant primary corneal fibroblasts.

A. Western blot analysis of TGFBIp in cell lysates (upper panel) and conditioned media (lower panel) of WT, HE, and HO cells. β-actin was used as a loading control. Molecular weight markers (in kDa) are indicated. B. Secretion of WT and mutant TGFBIp was inhibited by treatment with brefeldin A (BFA) and monensin (MON). C. TGFBIp co-localized with markers of cis-Golgi (upper panel), medial-Golgi (middle panel), and trans-Golgi (lower panel) in cortical cells. Representative confocal images of immunofluorescence staining of TGFBIp (green) with GM130, mannosidase II, and TGN38 (all red) are shown. Overlapping areas are displayed in yellow in the merged images. Bars = 25 μm. D. Secretion of mutant TGFBIp is delayed in GCD2 corneal fibroblasts. Corneal fibroblasts from a patient with a homozygous TGFBIp mutation and a WT control were pulse-labeled for 20 min using ³⁵S-cysteine and then incubated for 0, 15, 30, 60, 120, 180, and 240 min in unlabeled media before immunoprecipitation of TGFBIp from cell lysates and conditioned media. Phosphorimaging was performed after SDS-PAGE to detect TGFBIp. One representative experiment is shown. E. Quantitation of the experiment presented in D. Triplicate lysate samples were analyzed. *P < 0.05.