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. 2011 Dec 19;287(9):6441–6453. doi: 10.1074/jbc.M111.322024

FIGURE 4.

FIGURE 4.

Wild type PST and all PST mutants containing the PBR effectively bind to NCAM, whereas PST71 and PST62 that lack the PBR do not. V5-tagged NCAM was co-expressed with Myc-tagged PST or a PST mutant in COS-1 cells (A) or Lec2 CHO cells (B). After 18 h, cells were lysed and lysates were incubated with anti-Myc antibody to immunoprecipitate (IP) the PST proteins, followed by incubation with protein A-Sepharose beads for 1 h. Immunoprecipitates were subjected to SDS-PAGE and immunoblotting (IB) with anti-V5 antibody to detect co-immunoprecipitated NCAM (CoIP, IP: Anti-Myc antibody, IB: Anti-V5 antibody). To determine the relative expression levels of NCAM and PST proteins, an aliquot of lysate was removed prior to co-immunoprecipitation and subjected to SDS-PAGE and immunoblotting with anti-V5 antibody to detect NCAM (NCAM expression, Anti-V5 antibody) and anti-Myc antibody to detect the PST proteins (Enzyme expression, Anti-Myc antibody).