FIGURE 6.
The ability of PST H331K to inhibit the polysialylation of NCAM by endogenous SW2 PST is eliminated when Arg82 and Arg93 are replaced with alanine residues. V5-tagged NCAM and Myc-tagged PST were expressed in COS-1 cells without (lane 1) or with Myc-tagged PST H331K, PST H331K R82A, PST H331K R93A, or PST H331K R82A/R93A in a 1:1:6 ratio (lanes 2–5). After 18 h, cells were lysed and NCAM was immunoprecipitated (IP) using the anti-V5 antibody. Immunoprecipitated NCAM was subjected to SDS-PAGE and immunoblotting (IB) to analyze NCAM polysialylation (NCAM polysialylation, IP: Anti-V5 antibody, IB: OL.28 Anti-polySia antibody). Relative NCAM, PST, and PST mutant protein expression levels were assessed by removing an aliquot of cell lysate prior to immunoprecipitation and subjecting it to SDS-PAGE and immunoblotting with anti-V5 antibody (NCAM Expression, Anti-V5 antibody) or anti-Myc antibody (Enzyme Expression, Anti-Myc antibody). Note that enzyme expression reflects the expression levels of both wild type and mutant PST proteins.