A Novel Heterozygous Intronic Mutation in the FBN1 Gene Contributes to FBN1 RNA Missplicing Events in the Marfan Syndrome

Figure 4

Treatment of transfected cells with MG132 leads to detection of truncated proteins derived from mutant FBN1 constructs. (a) FLAG/Myc-tagged reference (FBN1-Ref; PL 1283) and mutant (FBN1-Mut; PL 1288) minigene plasmids (PL) were transfected into HeLa cells as indicated at the top of each lane. Lane 9: mock cells. Either the proteasome inhibitor MG132 or the DMSO vehicle was applied to transfected cells as indicated in Materials and Methods. Whole-cell extracts of transfected cells were analyzed by Western blotting with a mouse monoclonal anti-FLAG antibody. Black arrows: MW values (kDa) of the bands detected. (b) A diagram of the protein products as predicted by sequence analysis of the corresponding cDNAs. Deduced MW values (kDa) are also indicated. Dark grey box: partial inclusion of intron 22; F: FLAG epitope; M: Myc epitope; PTC: premature termination codon.

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