Fig. 4. Identification of GRP78 as the target antigen of NMO-rAbs ON-12-2-46 and ON-07-5-31. Identification of GRP78 as the target antigen of NMO-rAbs.

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Fig. 4. Identification of GRP78 as the target antigen of NMO-rAbs ON-12-2-46 and ON-07-5-31. Identification of GRP78 as the target antigen of NMO-rAbs ON-12-2-46 and ON-07-5-31. (A) Proteins isolated from crude membrane lysates of U87MG (glioblastoma) and OL (oligodendroglia) cells were separated by polyacrylamide gel electrophoresis and analyzed by Western blot. Multiple protein bands reacted with the NMO-rAbs. Unlabeled lanes contain molecular weight standards. (B) Schematic detailing the purification scheme (X-linker, cross-linker; DSS, disuccinimidyl suberate). (C) The protein eluted with DTT from rAb cross-linked to the U87MG cells using DTSSP/disuccinimidyl suberate (DSS) was subjected to polyacrylamide gel electrophoresis and immunoblot analysis (lane 1, molecular weight standards; lane 2, protein bound to ON-07-5-31 rAb; lane 3, protein bound to ON-12-2-46 rAb). The blots were probed with the indicated NMO-rAb. (D) Immunofluorescence labeling of U87MG glioblastoma cells with ON-07-5-31 rAb (10 μg/ml) and rabbit anti-GRP78 antisera shows colocalization of the two proteins (merge in yellow). Nuclei are counterstained with DAPI (blue). (E) ON-12-2-46 rAb (20 μg/ml; green channel) and rabbit anti-GRP78 antiserum (red channel) immunoreactivity on U87MG glioblastoma cells after 24 hours of treatment with 0.5 mM thapsigargin (TG) or DMSO (control). The merged image shows colocalization of the two proteins (yellow). (F) Immunoblot analysis of commercial recombinant GRP78 protein purified from transfected human embryonic kidney (HEK) 293 cells (lane 1) or bacteria (lane 2) and probed with rAb or rabbit anti-GRP78 serum demonstrates that NMO-rAbs ON-12-2-46 and ON-07-5-31 recognize GRP78. (G) Binding of ON-12-2-46 rAb (2 μg/ml) (green channel) to the U87MG glioblastoma cells in competition immunofluorescence assay is blocked in the presence of commercial recombinant GRP78 protein (5 μg). DAPI-stained nuclei are depicted in blue. Fumitaka Shimizu et al., Sci Transl Med 2017;9:eaai9111 Published by AAAS