Identification of the major FucT8 substrates. A: AAL lectin blot of control and FucT8 morphant embryo lysates resolved by SDS-PAGE. The morphant lysate has reduced AAL-positive proteins in the high molecular weight region (brackets). AAL lectin binding to these high molecular weight polypeptides is inhibited by excess fucose in both control and morphants samples, whereas the lower two bands are not and represent non-specific bands (indicated by the asterisk). B: Control and FucT8 morphant lysates were incubated with AAL-conjugated beads to precipitate the core fucosylated proteins, which were subsequently resolved by SDS-PAGE and subjected to AAL lectin blotting. AAL beads precipitate high molecular weight AAL-reactive glycoproteins that are not present in similarly treated morphant lysates (brackets). *, The same non-specific protein band seen in A. C: AAL-conjugated beads were used to pull down core fucosylated proteins from a HepG2 cell lysate, which were resolved by SDS-PAGE and probed with ApoB antibody. The anti-ApoB detects a large molecular mass polypeptide in HepG2 lysates that migrates above the 250-kDa marker, and anti-ApoB antibodies were able to immunoprecipitate a polypeptide of similar size. AAL beads were also able to precipitate an ApoB-reactive polypeptide of similar molecular weight, although not all ApoB was precipitated by AAL, since ApoB-reactive material remained in the AAL bead supernatant. D: Coomassie blue–stained gel of high molecular weight polypeptides, which correspond to the ApoB-containing, AAL-reactive bands in control embryos. The amount of high molecular weight ApoB polypeptides (brackets) appears to be somewhat reduced in FucT8 morphants.
|
