Anti rabbit antibodies

Mouse and rabbit IgG antibodies against SARS-CoV-2 spike or Ncap were measured by enzyme-linked immunosorbent assays (ELISAs) using precoated ELISA plates (IEQ-CoV-S-RBD-IgG and IEQ-CoV-N-IgG; RayBiotech), according to the manufacturer’s instructions, at RT. Briefly, plasma samples were diluted in sample buffer (RayBiotech), added to antigen-coated wells in triplicates, and incubated at RT for 2 h on a shaker (200 rpm). Commercially available antibody against spike (catalog number S1N-S58; Acro Biosystems) or Ncap (catalog number NUN-S47; Acro Biosystems) was used as a positive control. Plates were washed 3 times with wash buffer and incubated for 1 h at RT with HRP-conjugated goat anti-mouse secondary antibodies (dilution of 1:5,000) (catalog number 115-035-003; Jackson ImmunoResearch) or anti-rabbit antibodies (dilution of 1:5,000) (catalog number 111-035-003; Jackson ImmunoResearch) diluted in assay buffer (RayBiotech). After 3 washes, the plates were developed using the TMB substrate (RayBiotech). After a 15-min incubation, the reaction was stopped by the addition of a stop solution, and the absorbance at 450 nm was recorded using a BioTek Gen5 plate reader (Agilent). Endpoint titers were calculated as the dilution that emitted an optical density (OD) exceeding 4 times that for the PBS control group.

To analyze human AGXT2 transgene expression cell slides with primary aortic cells were fixed with 1:1 acetone-methanol solution for 10 min at 4 °C, washed 3 × 2 min with ice-cold PBS and blocked with Dako Protein Blocking solution for 20 min at room temperature. Afterwards, chamber slides were incubated with 1:100 rabbit polyclonal anti-FLAG antibodies (Sigma-Aldrich, St. Louis, MI, USA, Catalog #7425) for 2 h at 37 °C, washed 3 × 2 min with PBS and subsequently incubated with 1:250 anti-rabbit antibodies coupled with fluorescence dye (The Jackson Laboratory, Bar Harbor, ME, USA) at room temperature for 1 h. Finally, slides were washed 3 × 2 min with PBS, stained with 1:1000 DAPI and mounted with Moviol. To demonstrate the expression of human AGXT2-FLAG transgene in endothelial cells 1:100 rat anti-CD31 antibodies (Biolegend, San Diego, CA, USA, Catalog # 102401) (as marker of endothelial cells^{33 (link)}), and 1:250 anti-rat secondary antibodies (The Jackson Laboratory, Bar Harbor, ME, USA) were used. Double staining of CD31 and FLAG-tagged transgene was performed in the same way.

Mouse and rabbit IgG antibodies against SARS-CoV-2 spike or Ncap were measured by enzyme-linked immunosorbent assays (ELISAs) using precoated ELISA plates (IEQ-CoV-S-RBD-IgG and IEQ-CoV-N-IgG; RayBiotech), according to the manufacturer’s instructions, at RT. Briefly, plasma samples were diluted in sample buffer (RayBiotech), added to antigen-coated wells in triplicates, and incubated at RT for 2 h on a shaker (200 rpm). Commercially available antibody against spike (catalog number S1N-S58; Acro Biosystems) or Ncap (catalog number NUN-S47; Acro Biosystems) was used as a positive control. Plates were washed 3 times with wash buffer and incubated for 1 h at RT with HRP-conjugated goat anti-mouse secondary antibodies (dilution of 1:5,000) (catalog number 115-035-003; Jackson ImmunoResearch) or anti-rabbit antibodies (dilution of 1:5,000) (catalog number 111-035-003; Jackson ImmunoResearch) diluted in assay buffer (RayBiotech). After 3 washes, the plates were developed using the TMB substrate (RayBiotech). After a 15-min incubation, the reaction was stopped by the addition of a stop solution, and the absorbance at 450 nm was recorded using a BioTek Gen5 plate reader (Agilent). Endpoint titers were calculated as the dilution that emitted an optical density (OD) exceeding 4 times that for the PBS control group.

Cells were washed with DPBS (Lonza, Bornem, Belgium) and lysed with 30 µL/well of RIPA Lysis Buffer (Millipore, Darmstadt, Germany). They were supplemented with protease inhibitor (Complete Protease Inhibitor Cocktail Tablets, Roche, Basel, Switzerland) and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich, Hoeilaart, Belgium). Proteins (40 µg) were separated on an SDS-PAGE gel and transferred onto a nitrocellulose membrane. The following primary antibodies were used for protein detection: anti-HIF-1α mAb (D2U3T, Cell signaling, Leiden, The Netherlands), anti-CAIX (Novus, Abingdom, UK), and anti-β-actin monoclonal antibody (Sigma, Overijse, Belgium). Secondary antibodies included anti-rabbit antibodies (Jackson, Ely, UK). Tumors were harvested from euthanized mice and immediately frozen in isopentane. Tumors were reduced to small pieces without previous thawing. RIPA was added, and samples were sonicated and centrifuged. The supernatant was used for Western blotting. Proteins (80 µg) were separated on an SDS-PAGE gel and transferred onto a nitrocellulose membrane. Protein detection was performed as described above.