Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 4. 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 suber- ate). (C) The protein eluted with DTT from rAb cross-linked to the U87MG cells using DTSSP/disuccinimidyl suberate (DSS) was subjected to polyacrylamide gel elec- trophoresis 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 mg/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 mg/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 mg/ml) (green channel) to the U87MG glioblastoma cells in competition immunofluorescence assay is blocked in the presence of commercial recombinant GRP78 protein (5 mg). DAPI-stained nuclei are depicted in blue.

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: Isolation, Membrane, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight, Purification, Immunofluorescence, Labeling, Control, Recombinant, Transfection, Bacteria, Binding Assay, Staining

Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 5. Removal of GRP78-specific IgG from pooled NMO-IgG reduces NF-kB p65 nuclear translocation in BMECs. Numbers of cells positive for nuclear NF-kB p65 translocation were compared in cultures exposed to pooled NMO-IgG (200 and 150 mg/ml) immunoadsorbed against GRP78 or control SNAP25 protein. Data are normalized to control SNAP25-absorbed NMO IgG. Data are means ± SEM from five experiments in triplicate. Statistical significance was assessed by paired two-tailed t test (*P < 0.05).

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: Translocation Assay, Control, Two Tailed Test

Journal: Science translational medicine

Article Title: Glucose-regulated protein 78 autoantibody associates with blood-brain barrier disruption in neuromyelitis optica.

doi: 10.1126/scitranslmed.aai9111

Figure Lengend Snippet: Fig. 6. In vivo effects of ON-12-2-46 on mouse brain vasculature. (A) Experimental setup. IV, intravenously; IP, intraperitoneally. 3D images of 20-mm mouse coronal brain sections immunostained for human IgG (rAb ON-7-5-53) and AQP4 (B), human IgG (rAb ON-7-5-53) and mouse fibrinogen (C), or human IgG, mouse albumin, and AQP4 (D) after administration of murinized GRP78-specific NMO rAb ON-12-2-46 or control rAb in combination with human AQP4-specific rAb ON-7-5-53. (E) Mea- surement of the average diameter of vessels (mean ± SEM) in mice treated with GRP78-specific NMO rAb ON-12-2-46 (six mice) and control rAb (four mice) in two separate experiments. Vessels were quantified from control-rAb–injected (n = 35) and ON-12-2-46–injected (n = 31) 20× high-power fields, and the diameters were measured using AxioVision LE. P values were calculated by Mann-Whitney U test (***P < 0.0001).

Article Snippet: For Western blot verification, purified GRP78 protein [from OriGene (100 ng per well) and Novus Biologicals (0.5 mg per well)] was 9 of 12 SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E D ow nloaded from https://w w w .science.org on A pril 01, 2024 subjected to SDS–polyacrylamide gel electrophoresis, transferred to PVDF, incubated with 0.5% glutaraldehyde in PBS (15 min), with 0.25 M tris (pH 7.5) in PBS (15 min), washed three times in TBS (10 min), blocked, and then processed as described above.

Techniques: In Vivo, Control, Injection, MANN-WHITNEY

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 2 Effect of ACPAs and different protein molecules on the ratio of p-ERK1/2/ERK1/2, p-JNK/JNK and p-p38/p38 MAPKs in U937 cells. The concentration of different proteins were: non-specific human IgG (6 μg/ml), ACPAs (100 IU/ml), ACPA-depleted [ACPA(−) RA] sera (6 μg/ml), ACPAs (100 IU/ml)+anti-Toll-like receptor 4 blocking antibody (anti-TLR4, 10 μg/ml), ACPAs (100 IU/ml)+cit-GRP78 (10 μg/ml) or ACPAs (100 IU/ml)+GRP78 (10 μg/ml) for 24 h. The cells were lysed and both phosphorylated and native forms of three MAPKs were analyzed by Western blotting. a pERK1/2/ERK1/2 ratio, b p-JNK/JNK ratio, c p-p38/p38 ratio, and d A representative example to show the expression of phosphorylated and native forms of MAPKs after incubation with different proteins by Western blot; * denote p<0.05 compared to the non-specific human IgG

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Concentration Assay, Blocking Assay, Western Blot, Expressing, Incubation

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 4 Effect of ACPAs and different protein molecules on p-Akt/Akt and p-IKK-α/IKK-α ratio of U937 cells. U937 cells (1×106 cells/ml) were incubat- ed with non-specific human IgG (5 μg/ml), ACPAs (80 IU/ ml), ACPA(−) RA sera (5 μg/ ml), ACPAs (80 IU/ml)+anti- TLR4 (10 μg/ml), ACPAs (80 IU/ml)+cit-GRP78 (10 μg/ ml) for 24 h. The cells were lysed and both phosphorylated and native form of a Akt and b IKK-α were analyzed by West- ern blotting. * denote p<0.05 compared to the non-specific human IgG

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques:

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 5 Comparison of citrullinated (cit-GRP78) and native forms (GRP78) GRP78 molecules on the surface mem- brane extracts (150 μg) among U937 cells, normal human PBMCs, and RA PBMCs by Western blotting. The expres- sion of transferrin receptor (TfR) was used as an internal control. a The expression ratio of cit-GRP78/TfR among the three groups b The expression of GRP78/TfR among three groups c The expression ratio of cit-GRP78/GRP78 among three groups. d A representative example is shown

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Comparison, Western Blot, Control, Expressing

Journal: Journal of clinical immunology

Article Title: Anti-citrullinated protein antibodies activated ERK1/2 and JNK mitogen-activated protein kinases via binding to surface-expressed citrullinated GRP78 on mononuclear cells.

doi: 10.1007/s10875-012-9841-6

Figure Lengend Snippet: Fig. 6 A schematic diagram to indicate that the MAPK signaling pathways are transduced by ACPAs after binding to surface- expressed cit-GRP78 on RA PBMCs that leads to TNF-α production

Article Snippet: Recombinant GRP78 (Novus Biologicals, Littleton, CO, USA) was successfully citrullinated (cit-GRP78) after incubation of the molecules with 10 mM CaCl2 and peptidylarginine deiminase (Sigma-Aldrich) at a concentration of 20 IU per 1 mg GRP78 protein for 2 h.

Techniques: Protein-Protein interactions, Binding Assay

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: CDDO‐2P‐Im binds GRP78 and activates the PERK and IRE1α branches of the UPR in a dose‐dependent manner. (A) Cell lysate of RPMI‐8226 was incubated with DMSO or CDDO‐2P‐Im for 30 min. Lysates were then subjected to 0.025 mg·mL −1 pronase for 30 min at 4 °C, followed by SDS/PAGE and western blot using antibodies against GRP78 and GAPDH. CDDO‐2P‐Im protects GRP78 from pronase cleavage compared with DMSO control. (B) Thermal shift assay was performed on recombinant GRP78 treated with control (DMSO) or CDDO‐2P‐Im. Relative fluorescent units (RFU) were measured by a SpectraMax Paradigm plate reader. The melting temperature (Tm) has a shift of −1.9 °C with the treatment of CDDO‐2P‐Im. (C, D) ARH‐77 and RPMI‐8226 cells were incubated with CDDO‐2P‐Im for 6 h. Cells were lysed and samples were prepared for SDS/PAGE. Western blot of UPR signaling proteins and downstream targets were performed. (E) 5T33 tumors were extracted from mice 15 days after tumor injection. Mice were treated once with either vehicle or 24 mg·kg −1 CDDO‐2P‐Im for 12 h prior to extraction. Samples were immunoblotted for UPR proteins. (F) RNA‐Sequencing data for ATF6 regulated genes showed no differences between Control, 0.1 μ m CDDO‐2P‐Im, and 0.4 μ m CDDO‐2P‐Im in RPMI‐8226 cells. (G, H) Western blot of ATF6 in ARH‐77 and RPMI‐8226 show no changes at 6 h with the treatment of CDDO‐2P‐Im. Values were given as mean ± SD. Fragments Per Kilobase of transcript per Million mapped reads is abbreviated as FPKM. Western blot and DARTS data are representatives of at least three independent experiments for ARH‐77 cells and RPMI‐8226 cells. Thermal shift assay was performed three independent times and representative data was shown. Western blot of 5T33 tumors was performed once. RNA‐Sequencing was performed once. Student t ‐tests were performed to calculate P value. 2P‐Im, CDDO‐2P‐Im; CDDO, CDDO‐2P‐Im.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Incubation, SDS Page, Western Blot, Control, Thermal Shift Assay, Recombinant, Injection, Extraction, RNA Sequencing

Journal: Molecular Oncology

Article Title: The synthetic oleanane triterpenoid CDDO‐2P‐Im binds GRP78 / BiP to induce unfolded protein response‐mediated apoptosis in myeloma

doi: 10.1002/1878-0261.13447

Figure Lengend Snippet: Inhibition of the PERK‐ATF4‐CHOP arm of the UPR partially rescues CDDO‐2P‐Im‐induced apoptosis. (A) Cell lysate of WT and PERK KO RPMI‐8226 were probed for PERK protein by western blot. (B) WT and DDIT3 (CHOP) KO RPMI‐8226 were incubated with DMSO or CDDO‐2P‐Im for 6 h and lysed for protein analysis. Western blot of CHOP was performed to investigate the knockout of CHOP protein. (C) Cells were preincubated with DMSO or CDDO‐2P‐Im for 24 h and evaluated for cell viability by CellTiter‐Glo™. (D) Cells were preincubated with DMSO or 0.25 μ m ISRIB for 3 h before treating with DMSO or CDDO‐2P‐Im for an additional 16 h. Cells were then measured for cell viability by CellTiter‐Glo™. Controls were cells that were not treated with CDDO‐2P‐im but were treated with DMSO or ISRIB where appropriate. (E–H) PERK KO and WT RPMI‐8226 cells or ARH‐77 cells pretreated with ISRIB for 3 h were incubated with DMSO control or 0.4 μ m CDDO‐2P‐Im for 6 h. (E, F) Cells were extracted for RNA and qRT‐PCR was performed to investigate changes in the UPR. (G, H) In another round of experiments, cells were also extracted for protein to confirm such changes in the UPR. Values were given as mean ± SD. Student t ‐tests were performed to calculate P value. * P < 0.05 and ** P < 0.01, compared with control. Cell viability data are representative of three independent experiments. Western blots and qRT‐PCR data are representatives of two independent experiments. (I) We provide a working model of the actions of CDDO‐2P‐Im in cancer cells. At low concentrations (panel A), 2P‐Im binds and inhibits KEAP1, an adaptor protein for ubiquitin ligase that negatively regulates Nrf2 levels. Nrf2 will translocate to the nucleus and dimerize with small Maf proteins (sMaf) to activate the transcription of Nrf2 target genes. At higher concentrations (panel B), 2P‐Im binds GRP78/BiP, resulting in the activation of the UPR. GRP78 dissociates from its binding partners leading to the phosphorylation of PERK and IRE1α and activation of these branches of the UPR. The transcription factors, CHOP and XBP1, will cause UPR‐associated gene expression changes which when prolonged will lead to apoptosis. Interestingly, XBP1 is known to increase the expression of HRD1, a negative regulator of Nrf2, and independent of KEAP1. 2P/2P‐Im, CDDO‐2P‐Im; ISRIB, integrated stress response inhibitor; WT, wild‐type; KO, knockout.

Article Snippet: 10 μ m of recombinant GRP78 protein (PROTP11021; Boster Biological Technology, Pleasanton, CA, USA) was incubated with 150 μ m 2P‐Im controlled with 1% DMSO, and thermal shift assay was performed as previously described [ ].

Techniques: Inhibition, Western Blot, Incubation, Knock-Out, Control, Quantitative RT-PCR, Ubiquitin Proteomics, Activation Assay, Binding Assay, Phospho-proteomics, Gene Expression, Expressing