Fig. 4

Consequences of the 10 bp insertion at the transcript and protein level. (A) Duplex PCR suggests a 10 bp insertion results in partial nonsense-mediated decay. RT-PCR products using primers for VWA1 and ACTB using RNA from proband in Family 11 (P) compared to control fibroblasts (C2, C2). The ratio of VWA1 to ACTB was reduced in the patient but was restored when patient fibroblasts were grown in presence of the nonsense-mediated decay inhibitor cycloheximide (CHX). (B) Immunoblotting indicated detectable levels of VWA1 (WARP) in human healthy control dermal fibroblasts; although the secreted VWA1 was not able to incorporated into ECM (cell layer), it was detectable in the conditioned medium. In contrast, no detectable VWA1 was seen in the patient’s dermal fibroblast in either the conditioned medium or the cell layer. A high level of VWA1 was detected in mouse sciatic nerve extraction (m-nerve). In the primary culture of mouse sciatic nerve (mnf), the VWA1 was detectable in its cell layer (cytoplasm and ECM) but not in the conditioned medium, indicating the secreted VWA1 was efficiently deposited as ECM. Tubulin and fibronectin (FN) used as loading controls. GuCHL = guanidine hydrochloride extraction.