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In order to fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in-depth knowledge of versican’s biosynthetic processing is necessary. We have recently shown that b-catenin/T-cell factor (TCF) complex formation at the versican promoter site is essential for activation of versican transcription. The transcriptional activator b-catenin is the key mediator of the canonical Wnt signaling pathway. However, b-catenin does not itself bind DNA and thus functions via interaction with TCF/Lymphoid-enhancing factor (LEF) transcription factors.
There is always a nominal percentage of protein that is secreted in its unmodified state, and this unmodified protein migrates faster on SDS-PAGE, by virtue of its lower molecular mass. Western blot analysis is used to determine the percent of glycosylation of the reporter protein, by calculating the ratio of the glycosylated protein (higher MW) over the unmodified (lower MW) isoform in the same lane (see Fig. 3b). 2. Important controls for Western blot analysis of glycoproteins— to verify that the unmodified form of a protein doublet is the lower MW protein species, the reporter construct is transfected in a parallel set of cells with and without RNAi-treatment to knock down the glycosylation machinery, to demonstrate the mobility of the same peptide sequence in the absence of glycosylation (see Fig.
And Schwartz, N. B. , Dev. Biol. 14, 169–198. Krueger, R. , Fields, T. , Mensch, J. , and Schwartz, N. B. (1990) Chick cartilage chondroitin sulfate proteoglycan core protein II. Nucleotide sequence of cDNA clone and localization of the S103L epitope, J Biol Chem 265, 12088–12097. , Domowicz, M. , and Schwartz, N. B. (1996) S103L reactive chondroitin sulfate proteoglycan (aggrecan) mRNA expressed in developing chick brain and cartilage is encoded by a single gene, Mol. Brain Res. 36, 309–321.