Anti β actin

Protein extraction, quantification, separation in gel electrophoresis and transfer were performed using standard procedure [3 (link)]. Nitrocellulose membranes were blocked in Odyssey-TM Blocking Buffer (927–50003, LICOR) for 1 h and further incubated overnight at 4 °C with the following primary antibodies diluted in OdysseyTM Blocking Buffer: anti-RARS2 (1:16000, ab230274, Abcam), Anti-β Actin (1:10000, AM4302, Invitrogen). After three washes with 0.2% PBST, the membranes were then incubated for 1 h at room temperature with IRDye-coupled (1:10000, 925–68070, 926–32211, LICOR) secondary antibodies. After three washes with 0.2% PBST, the membranes were processed with Odyssey CLx imaging system (LICOR) and the quantification analysis was performed with Image Studio Lite Ver 5.2, with the default parameters and using β Actin for normalization. Detection and quantification of RARS2 protein level in patient and control was repeated in two independent experiments with comparable results (Supplementary Fig. 1D-F).

Renal cortex was homogenized and extracted in a cold buffer containing 0.1 mol/l Tris (hydroxymethyl) aminomethane HCl. The tissue extracts were then partially purified by ethanol extraction. Proteins from GENs or mesangial cells were isolated from using RIPA buffer (Thermo Scientific, USA). 50 μg of protein samples were isolated in 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, USA) by electroblotting. The membranes were blocked in 5% non-fat dried milk for 60 min at room temperature. For the detection of RhoA, the membrane and cytoplasm proteins were extracted from mesangial cells using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology), then membrane and cytosolic proteins were separated on SDS-PAGE. The membranes were probed with first primary antibody anti-ETBR (Abcam, USA), anti-ETAR (Abcam, USA), anti-p-p65 (Invitrogen, USA), anti-p65 (Invitrogen, USA), anti-CTGF (Abcam, USA), anti-RhoA (Abcam, USA), anti-collagen IV (Abcam, USA), anti-Fibronectin (Abcam, USA), anti-p21 (Abcam, USA) and anti-β-actin (Invitrogen, USA) and incubated at 4°C overnight. After washing with PBST, membrane was cultivated with secondary antibody for 60 min at room temperature. β-actin was used as internal control.

For flow cytometry, anti-B220-PE/Cy7 (RA3-6B2), anti-CD19-BV510 (GD5), anti-IgD-PE (11-26c.2a), anti-CD23-APC/Cy7 (B3B4), anti-IgM-APC (RMM-1), anti-CD21-PerCP/Cy5.5 (7E9), and anti-CD24-PE (30-F1) were purchased from Biolegend. For magnetic cell separation, biotinylated anti-IgM (RMM-1), anti-B220 (RA3-6B2), anti-Gr1 (RB6-8C5), anti-CD23 (B3B4), and anti-CD3e antibody (145-2C11) were purchased from BD Pharmingen. For microscopy, anti-α-smooth muscle actin (ab5694, Abcam), anti-IgM-Cy3 (EMD Millipore), anti-IgD-FITC (11-26c, Invitrogen), anti-MARCO-FITC (Bio-rad), and anti-B220 (RA3-6B2, eBioscience) were used. For western blot, anti-NFkB2, anti-p-p38 (T180/Y182), anti-pAkt (S473) from Cell Signaling Technology, anti-β-actin (Invitrogen), anti-BAFFR and anti-ST6Gal-1 (R&D Biosystems), and anti-pTyr (EMD Millipore) were used.

WB was performed as previously described [49 (link)]. Both anti-IGF2BP3 (Cat# A4444) and anti-Flag (Cat# AE063) antibodies were purchased from Abclonal (Wuhan, China). Anti-ARHGAP11A (Cat# PA5-101840), anti-PD-L1 (Cat# M033179), and anti-β-actin (Cat# AC006) antibodies were purchased from Invitrogen, Abmart (Shanghai, China), and Sigma-Aldrich, respectively. The protein level was normalized with β-actin.