Ab108252

Rat cardiac tissue (25 mg) was homogenized and mixed in 1500 µL of RIPA buffer with a protease inhibitor tablet (1 tablet per 10 mL) (P/N 11697498001, Sigma Aldrich, St. Louis, MO, USA). The supernatants were taken after centrifugation for protein quantification with the Qubit Protein Reagent. The same amount of protein (128 µg) per lane was separated electrophoretically by Tris-Glycine (4–12%) Gel and transferred to a PVDF membrane. The membranes were incubated with a primary Ab, ACE (rabbit monoclonal EPR22971-247 to ACE1, ab25422, Abcam1:1000), and ACE2 (rabbit monoclonal EPR4435(2) to ACE2, ab108252, Abcam1:1000), AT1R (Rabbit monoclonal EPR3873 to AT1R, ab124734, Abcam1:1000), AT2R (rabbit monoclonal EPR3876 to AT2R, ab92445, Abcam1:1000), and β-actin (mouse monoclonal AC-15 to β- actin, ab6276, Abcam1:1000) overnight in 4 °C. After washing four times for 10 min in TBS containing 0.1% Tween 20, the membranes were incubated with rabbit anti-mouse IgG with HRP secondary Ab (NBP1-73435, Novus 1:10,000) for 1 h at room temperature. Membranes were washed, incubated with Radiance Q Chemiluminescent substrate (Azure Biosystems, Dublin, CA, USA), and imaged by Azure biosystems c600 for 5 s⁻¹ min. The density of a specific band was quantified using ImageJ software.

For IHC, an automated IHC stainer BOND-III (Leica Biosystems) was used. Briefly, 4 μm sections of paraffin-embedded kidneys were submitted for appropriate antigen retrieval. Then, sections were incubated with the following antibodies: rabbit monoclonal anti-ACE2 antibody (Abcam, ab108252, 1:100), rabbit monoclonal anti–cleaved caspase-3 antibody (Cell Signaling Technology, 9664, 1:200), rabbit polyclonal anti–recombinant nucleoprotein from SARS-CoV antibody (a gift from Nicolas Escriou, Institut Pasteur, Paris, France), and rabbit polyclonal SARS-CoV-2 anti-nucleoprotein (Novusbio, NB100-56576, 1:500). ACE2 expression was evaluated by image quantification using the Integrated Density program of ImageJ software (NIH) on whole-kidney sections (×200). This measurement integrates the product of area and the mean intensity value above a specific threshold. This estimates the amount of the strength of the expression of a specific epitope.
Immunofluorescence was performed on frozen kidney biopsies using antibodies targeting the heavy chains of immunoglobulins (IgA, IgG, IgM), kappa and lambda light chains, complement (C3, C1q), and fibrinogen using the automated stainer BOND-III (Leica Biosystems). Immunofluorescence staining was performed on 26 of 32 biopsies.

Severe acute respiratory syndrome coronavirus 2 nucleoprotein was co-stained with ACE2 and TMPRSS2, respectively. Anti-SARS-CoV-2 nucleoprotein antibody (Cat No. 40143-T62; Sino Biological), anti-TMPRSS2 antibody (ab109131; Abcam), and anti-ACE2 antibody (ab108252; Abcam) were used in this assay.

Livers were dissected and subsequently frozen in liquid nitrogen. The tissue of each liver was homogenized in a buffer and centrifuged at 14,000×g. Protein (65 μg) from the supernatant of each sample was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked in TBST buffer containing 10% non-fat milk for 1 h at room temperature. Immunoblotting was performed by incubating the blocked membrane overnight at 4 °C with a monoclonal mouse leptin receptor antibody (Gene Tex/GTX37636, Irvine, CA, USA), Sirt1 antibody (cell signaling /#9475, Danver, MA, USA), ACE2 antibody (abcam/ab108252, Cambridge, MA, USA), and malondialdehyde (MDA) antibody (abcam/ab27642, Cambridge, MA, USA). The membranes were then incubated with secondary HRP conjugated anti-rabbit antibody (1:5000; Jackson Immuno Research, West Grove, PA USA) or anti-mouse antibody (1:10,000; Jackson Immuno Research, West Grove, PA USA) for 1 h at room temperature. Western blots were visualized using an ECL kit (Perlcin Elmer In./NEL 105001EA, Boston, MA, USA).