Materials and Methods Supplementary Information
cDNA and Plasmid constructs
pcDNA3.1 HA podocin was generated by subcloning HA-tagged human podocin cDNA (GenBank: NM_014625) cloned into pcDNA3.1 vector. For GST fusion constructs, three C-terminal fragments of podocin amino acid (AA) 262-383 (C1), AA 202-383 (C2) and AA 137-383 (C3), and those for CAR corresponding to AA 301-365 (C1), AA 281-365 (C2) and AA 261-365 (C3) were amplified from human podocin and CAR cDNA (GenBank: Y07593), respectively. These fragments were inserted into pGEX 4T-1 vector using EcoRI (podocin), or XhoI and BamHI (CAR) sites. For CAR-EGFP construct, the N-terminal CAR fragment encompassing the transmembrane domain (N-TM-C1, AA 1-285), the C-terminal part of CAR (C2, AA 286-365), or EGFP sequence (AA 1-239) were PCR-amplified from human CAR cDNA or pEGFP-N3 and the 3 fragments were ligated into pZeoSV2 vector. A human transferrin receptor (hTfR) deletion mutant (hTfR del 5-41) lacking AA 5-41 was constructed as previously reported (17).
Cell culture and antibodies
Mouse L was a generous gift from Dr. A. Nagafuchi (Kumamoto University). Primary cultured podocytes were prepared as described (33). The rabbit anti-podocin polyclonal (p) Ab was generated against a synthetic peptide comprising the 17 carboxy-terminal amino acids of human podocin. Rabbit anti-CAR pAb and gp135 were kindly supplied from Drs. Y. Hotta and R. Kuwano (Niigata University), and T.
Tsukamoto (Kobe University), respectively. Other antibodies were obtained from the following resources: mouse anti-CAR mAb (clone RmcB) (Upstate), rabbit anti-CAR (H-300) pAb (Santa Cruz Biotechnology Inc.), rabbit anti-ZO-1 pAb (Zymed Lab.), mouse anti-V5 mAb (Invitrogen Corp.), mouse anti-HA (clone 12CA5) mAb (Roche Diagnostics Corp.), rabbit anti-caveolin-1 pAb (BD Biosciences-Pharmingen), sheep
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anti-mouse IgG pAb (Sigma), and Alexa Fluor 633 phalloidin (Invitrogen Corp.). Cells were stained with antibodies to podocin (1:800 dilution), V5 (1:200 dilution), and ZO-1 (1:200).
Supplementary Figures
Supplementary Figure 1 Protein expression and proteinuria profiles of PAN
treated rats. (A) Western blot analysis of CAR and podocin. Lysates from isolated rat glomeruli were analyzed before and after PAN injections. (B) Profile for urinary albumin excretion in rats with PAN (mean ± SE, n = 5), P < 0.05.
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