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3 protocols using dibutyl camp

1

Modulating Fibrosis in Proximal Tubular Cells

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NRK-52E, the rat proximal tubular cell line, was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 supplemented with 10% fetal bovine serum. Cells were then cultured in fibronectin pre-coated flasks until they reached 70–80% confluence. IMD was over-expressed in NRK-52E by gene transfer with pcDNA3.1-IMD as we previously described [14 (link)]. Cell fibrosis model was induced by stimulating with recombinant human TGF-β1 (rhTGF-β1, 10 ng/ml, Gibco, Carlsbad, CA) for 72 h. The cells were randomly allocated into 1 of the following six groups: control (untreated cells); TGF-β1 (cells were stimulated with rhTGF-β1); TGF-β1 + empty vector (cells were transfected with the control plasmid pcDNA3.1 and stimulated with rhTGF-β1); TGF-β1 + IMD (cells were transfected with pcDNA3.1-IMD and stimulated with rhTGF-β1); TGF-β1 + dibutyl-cAMP [cells were stimulated with rhTGF-β1 plus cAMP analog dibutyl-cAMP (10−3 mol/l, Sigma-Aldrich, St. Louis, MO)]; and TGF-β1 + IMD + H-89 [cells were transfected with pcDNA3.1-IMD and stimulated with rhTGF-β1 plus PKA inhibitor H-89 (10−5 mol/l, Sigma-Aldrich, St. Louis, MO)].
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2

Purification and Characterization of Immunotoxins

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HA22 (27 (link)), LMB-11 (18 (link)), LMB-31 and SS1P (28 (link), 29 (link)) were purified as described (30 (link)). Immunotoxin LMB-31, an inactive form of LMB-11, was generated by site directed mutagenesis changing E553 to D553 in the PE fragment. RG7787 (huSS1(Fab)-LR-GGS-LO10-PE24) was supplied by Roche Innovation Center Penzberg, Germany under a Cooperative Research and Development Agreement (31 (link)).
Antibodies detecting p-S6K1(T389), S6K1, p-CREB(S133)/ATF-1, p-rpS6(S240/244), rpS6, p-GSK3β (S9), GSK3β, Bcl2, PARP, cleaved caspase 3, Bax, Bcl-xL, and MCL1 were purchased from Cell Signaling (Danvers, MA); H89 from Selleck (Houston, TX); 8-Br-cAMP, Dibutyl-cAMP and anti-β-Tubulin from Sigma (St. Louis, MO); anti-Actin from Abcam (Cambridge, MA); and inhibitors PKI-myristoylated (14 (link)–22 (link)) amide and S6K1 inhibitor PF4708671 from Tocris Sciences (Pittsburgh, PA).
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3

Investigating Vascular Inflammation Pathways

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Human CGRP, Human CGRP8–37, Ang II, N‐acetyl‐L cysteine (NAC), apocynin, PP2, H‐89 and dibutyl‐cAMP were purchased from Sigma. Niclosamide was purchased from Selleck Chemicals; anti‐phospho‐Src (Tyr416), anti‐phospho‐STAT3 (Tyr705), Src and STAT3 antibodies, from Cell Signaling Technology Inc; antibodies for p47phox and GAPDH, and all secondary antibodies, from Santa Cruz Biotechnology; antibodies of RAMP1, CRLR and ATP1a1, from Santa Cruz Biotechnology; RCP antibodies, from NOVUS Institute of Biotechnology; Alzet mini‐osmotic pumps (Alzet model 1004), from DURECT Corp; and the Src (B0107) and STAT3 (F0806) ORF cDNA clones, from GeneCopoeia. Other chemicals and reagents were of analytical grade.
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