FIGURE 8.

Mutation of O-linked glycosylation site (T648A) does not affect cell surface translocation of GRP78. A, schematic diagram of O-linked glycosylation sites predicted by the Net OGly 3.1 program for human GRP78. The threshold line of glycosylation potential is indicated as a black boldface line. The amino acid sequence position is shown below. B, the amino acid sequences surrounding the putative O-linked glycosylation site at aa 648 of human GRP78, with the mutated site indicated in boldface type. C, detection of surface and total intracellular F-GRP78 (wild type and mutant) in MCF7, HeLa, and 293T cells. β-Actin served as the loading control for the lysate input. Representative Western blots are shown. D, after quantitation of protein band intensity, the percentages of cell surface F-GRP78 (sF-GRP78; wild type (WT) and mutant) in the different cell lines were calculated and are presented. The experiments were repeated 3–4 times. The S.D. is shown.