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Submitted references Identification of two major autoantigens negatively regulating endothelial activation in Takayasu arteritis.

Mutoh T, Shirai T, Ishii T, Shirota Y, Fujishima F, Takahashi F, Kakuta Y, Kanazawa Y, Masamune A, Saiki Y, Harigae H, Fujii H
Nature communications 2020 Mar 9;11(1):1253

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of human EPCR in human kidney with EPCR Recombinant Rabbit Monoclonal Antibody (41) (Product # MA5-29505, 1:200, formalin-fixed paraffin embedded sections).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical staining of human EPCR in human liver with EPCR Recombinant Rabbit Monoclonal Antibody (41) (Product # MA5-29505, 1:200, formalin-fixed paraffin embedded sections).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 2 Identification of endothelial protein C receptor (EPCR) and scavenger receptor class B type 1 (SR-BI) as endothelial autoantigens in Takayasu arteritis. a HUVEC cDNA fragments inserted into the genomic DNA of C1 and C3 clones established with U10-4 IgG were amplified, and PCR products were electrophoresed on a 0.8% agarose gel. b DNA sequencing was performed for the PCR products obtained around 2000 bp for C1, followed by BLAST analysis. c C1 (left) and C3 (right) were stained with PE-conjugated isotype control or PE-conjugated anti-human EPCR antibody and analyzed with flow cytometry. d The expression vector EPCR-IRES-GFP was transfected into YB 2/0 cells, and the cells were stained with 0.5 mg/mL control IgG or U10-4 IgG, followed by incubation with secondary antibody and flow cytometry analysis. e Inhibition tests for binding activities to YB2/0 cells overexpressing EPCR were performed using 0.5 mg/mL U10-4 IgG with soluble recombinant EPCR at the indicated concentrations. f Western blotting of recombinant EPCR proteins was performed, and they were stained with control serum, U10-4 serum, or anti-human EPCR antibody, followed by secondary antibodies. g HUVEC cDNA fragments inserted into the genomic DNA of C6 clones established with W10-59 IgG and C7 by using G10-43 IgG were amplified, and PCR products were electrophoresed on a 0.8% agarose gel. h DNA sequencing was performed for the PCR products obtained around 3000 bp for C7, followed by BLAST analys

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Blocking of anti-inflammatory activities of EPCR and SR-BI by autoantibodies in Takayasu arteritis. a , b HUVECs were treated with or without 10 µg/mL APC for 13 h and stimulated with 100 pg/mL TNF-alpha for 5 h. The expression of adhesion molecules, including E-selectin, VCAM-1, and ICAM-1, was analyzed with flow cytometry. Representative histograms (left) and the summary graph (right, n = 3) are shown in a . The mRNA expression level was measured by quantitative PCR in b . GAPDH was used as the internal control. c HUVECs were incubated with the isotype or 10 µg/mL anti-EPCR antibody for 1 h and treated with or without 10 µg/mL APC for 13 h. Then, cells were stimulated with 100 pg/mL TNF-alpha for 5 h. The expression of adhesion molecules was analyzed with flow cytometry. d HUVECs were incubated with or without IgG for 1 h. IgG included 10 µg/mL anti-EPCR antibody, 2.56 mg/mL control IgG, 2.56 mg/mL IgG from an AECA-negative TAK sample (L11-05), or 2.56 mg/mL IgG from anti-EPCR-positive TAK AECA sample (J11-14). The cells were subsequently treated as described above, and the expression of adhesion molecules was analyzed. e HUVECs were treated with or without 1 mg/mL high-density lipoprotein (HDL) for 16 h and stimulated with 100 pg/mL TNF-alpha for 5 h. Adhesion molecules were analyzed by flow cytometry; the summary graph is shown ( n = 5). f HUVECs were incubated with or without IgG for 1 h. IgG included 10 µg/mL anti-SR-B

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