Turbo transfer

Following incubation with acP, purified proteins were separated by SDS-PAGE. The gel and the nitrocellulose membrane were equilibrated for 15 minutes in transfer buffer containing 25 mM Tris, 0.192 M glycine, 20% (v/v) methanol, and 0.1% (w/v) SDS. Proteins were transferred onto the membrane for 30 min using a Biorad TurboTransfer. The blot was blocked with 5% (w/v) milk prepared in TBST for 1 hour at room temperature. The blot was washed with TBST for 5 min. An anti-acetylated lysine-containing peptide (Cell Signaling 9441) was used at a 1∶1000 dilution in 5% BSA in TBST at 4°C overnight. The blot was washed 3 times for 5 min each with TBST and then incubated with HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling 7074S) at a 1∶1000 dilution in 5% milk in TBST for 1 hour at room temperature. The blot was washed 3 times for 5 min each with TBST and exposed using 20X LumiGLO^R Reagent and Peroxide (Cell Signaling 7003). Quantification of acetylation in terms of average pixel density was performed using AlphaView.

Around 1 × 10⁶ cells were collected and lysed in Alfa Aesar Nonidet 40 (NP-40; 20 mM Tris–Hcl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF). Cell lysates were fractionated on SDS-PAGE pre-cast gels (BioRad) and transferred to a PVDF membrane by TurboTransfer (BioRad). Immunoblotting assays were performed with the following primary antibodies (1:1,000): anti-ACE2 (ProSci, cat#3217), anti-DYRK1A (Abcam, cat#ab259869), anti-FLAG (Sigma-Aldrich, cat#F3165), anti-DPP4 (R&D, cat#AF1180), anti-GAPDH (BioLegend, cat#649202), and anti-Lamin B1 (BioLegend, cat# 869801). Proteins were visualized with goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:5,000) diluted in 2% Omniblot milk (AmericanBio) in 1× TBST using a chemiluminescence detection system (BioRad ChemiDoc MP).

Renal cortices were homogenized using a Next Advance Bullet Blender 24 (Averill Park, NY, USA) at 4 °C, with 1.00 and 2.00 mm beads at speed 8 for 4 min in a RIPA extraction buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS) at pH 7.4, containing a protease/phosphatase inhibitor cocktail (#5872, Cell Signaling Technology, Danvers, MA, USA). The protein content was determined using a BCA Protein Assay Kit (Pierce-Thermo Fisher Scientific, Melbourne, Australia). Equal amounts of the protein were separated on 4–20% Mini-Protean TGX Stain-Free gels (Bio-Rad Laboratories, Gladesville, NSW, Australia). Gels were then transferred to a polyvinylidene fluoride (PVDF) membrane on Turbo-Transfer (Bio-Rad laboratories, Gladesville, NSW, Australia). Membranes were blocked in 5% skim milk-Tris buffered saline, 0.1% Tween-20 (TBS-T) for 1 h before blotting with a MG-H1 antibody (hm5017; Hycultec GmbH, Beutelsbach, Germany) overnight at 4 °C. After washing and incubating with an HRP-conjugated polyclonal anti-mouse secondary antibody (P0260, Agilent Technologies, Mulgrave, VIC, Australia), membranes were visualized using a ChemiDoc™ Touch Imaging System (Bio-Rad laboratories, Gladesville, NSW, Australia). Samples were normalized by whole-lane stain free quantification to verify equal loading between lanes.