A3415

Equal amounts of 30 μg proteins from total extracts obtained by podocytes and RPTECs were separated by 12–15% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% BSA in tris-buffered saline (TBS) supplemented with 0.05% Tween-20 (Sigma-Aldrich), membranes were incubated with mouse anti-tubulin (1:2000; T9026, Sigma-Aldrich) or rabbit anti-SOD2 acetylated at lysine 68 (SOD2^AcK68, 1:1000; ab137037, Abcam) and, on the same membrane, sheep anti-total SOD2 (1:1000; 574596, Calbiochem). The signals were visualized on an Odyssey^® FC Imaging System (LiCor, Lincoln, NE, USA) by infrared (IR) fluorescence using secondary goat anti-rabbit IRDye 800CW antibody (FE30926211, LiCor), goat anti-mouse IRDye 680LT (FE3680200, LiCor) or donkey anti-sheep HRP antibody (1:20,000; A3415, Sigma-Aldrich), as appropriate. Bands were quantified by densitometry using the Image Studio Lite ver 5.0 software (LI-COR Biotechnology, Lincoln, NE, USA). SOD2 acetylation was expressed as the ratio between SOD2^AcK68 and total SOD2. Tubulin expression was normalized by Ponceau S staining.

Protein samples were resolved by SDS–PAGE in NuPAGE Novex 4–12% Bis-Tris gels (Thermo Fisher Scientific, NP0321 and WG1402A) either with NuPAGE MOPS SDS buffer (Thermo Fisher Scientific, NP0001) or NuPAGE MES SDS buffer (Thermo Fisher Scientific, NP0002), or with NuPAGE Novex 3–8% Tris–Acetate gels (Thermo Fisher Scientific, EA0375BOX and WG1602BOX) using NuPAGE Tris-Acetate SDS buffer (Thermo Fisher Scientific, LA0041). Resolved proteins were either stained with InstantBlue (Expedeon, ISB1L) or were transferred to a nitrocellulose membrane with the iBlot2 Dry Transfer System (Invitrogen, IB21001S). Antibodies used for protein detection in this study are described in Supplementary file 1. Conjugates to horseradish peroxidase of anti-sheep IgG from donkey (Sigma-Aldrich, A3415), or anti-mouse IgG from goat (Sigma-Aldrich, A4416) were used as secondary antibodies before the detection of chemiluminescent signals on Hyperfilm ECL (cytiva, 28906837, 28906839) with ECL Western Blotting Detection Reagent (cytiva, RPN2124).

Cell lysates were prepared on ice with RIPA buffer (R0278; Sigma-Aldrich) substituted with protease and phosphatase inhibitors (1861284; Thermo Fisher Scientific). Proteins were separated by 4–12% gradient SDS-PAGE in Tris-HCl buffer and transferred to nitrocellulose membranes. Equal transfer was verified with Ponceau Red stain (P7170-1L; Sigma-Aldrich). Membranes were blocked in 5% nonfat milk (42590.10; Serva) and probed with primary antibodies overnight at 4°C. Anti-P21 (2947S) was obtained from Cell Signaling Technology. Anti-CD47 (AF4670) antibody was purchased from R&D Systems. Anti-CD22 (ab218340) and anti-CD24 (ab179821) were obtained from Abcam. As a loading control, anti-GAPDH (2118) antibody was used from Cell Signaling Technology. Membranes were washed and goat anti-rabbit IgG-horseradish peroxidase (1:2,500; RPN4301; Amersham), goat anti-mouse IgG-horseradish peroxidase (1:1,500; A8924; Sigma-Aldrich) or donkey anti-sheep IgG-horseradish peroxidase (1:4,000; A3415; Sigma-Aldrich) were used as secondary antibody. Membranes were developed using chemiluminescence reagents (NEL104001EA; Perkin Elmer) and the X-Stella system (Reytest Isotope Messgeraete GmbH). Image analysis was performed using the AIDA Imaging analyzer (Reytest Isotope Messgeraete GmbH).