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2 protocols using alzet mini osmotic pumps alzet model 1004

1

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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2

Macrophage Polarization and Inflammatory Signaling

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Synthetic human IMD1–53, rat IMD17–47 and AngII were from Phoenix Pharmaceuticals (Belmont, CA, USA). Alzet Mini-osmotic pumps (Alzet model 1004) were from DURECT Corp. (Cupertino, CA, USA). DAPT, LPS, IFN-γ, IL-4, PI3K/Akt inhibitor LY294002, cAMP/PKA inhibitor H89 and cGMP/PKG inhibitor Ly83583 were from Sigma (MO, USA). Primary antibodies for Notch1 (ab52627), hes1 (ab71559), NICD (ab8925), iNOS (ab3523), NLRP3 (ab214185), caspase-1 (ab1872), CD68 (ab955), CD16 (ab203883), Arg1 (ab91279), CD206 (ab64693, ADAM10 (ab1997), ADAM17 (ab2051), IL-1β (ab2105), IL-10 (ab9969) and IL-18 (ab191860) were from Abcam (Cambridge, UK). Primary antibodies for p-Akt (Ser473) (4060) and Akt (9272) were from Cell Signaling Technology(CST, USA). Primary antibodies for β-actin (sc-1616), GAPDH (sc-25778), ASC (sc-514414), and all secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Other chemicals and reagents were of analytical grade. Mouse macrophage line Raw264.7 were bought from American Type Culture Collection (ATCC, USA).
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