Ro 32 0432

Live cells were incubated with 5 µM PKC inhibitor Ro-32-0432 (Sigma-Aldrich, 557525) overnight. CD4 expression was analyzed by flow cytometry (antibody clone RM4-5).

Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC. HUVECs were cultured with M199 basal medium supplemented with low-serum growth supplement and penicillin (50 IU/ml)-streptomycin (50 μg/ml). trypsin-EDTA was used to passage cells. M199 and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). Low-serum growth supplement was purchased from Cascade (Portland, OR, USA). Additionally, 5,58,6,68-tetraethylbenzimidazolcarbocyanine iodide (JC-1) were purchased from BioVision (Palo Alto, CA, USA). LY294002, dihydroethidium (DHE), Apocynin, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), DCHC, AICAR, sodium nitroprusside (SNP), Ro-320432, penicillin and streptomycin were all purchased from Sigma (St. Louis, MO, USA). Anti-β-actin, anti-AMPK, anti-AMPK-α, anti-AKT, anti-phospho AKT, anti-LOX-1, anti-SIRT1, anti-eNOS were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). HRP-conjugated anti-rabbit secondary antibodies were purchased from Transduction Laboratories (CA, USA). (TUNEL) staining kit was obtained from Boehringer Mannheim (Mannheim, Germany) was purchased from Selleckchem. Fura-2 AM and the EnzChek caspase 3 assay kit were purchased from Molecular Probes (Eugene, OR). SOD activity assay kit was obtained from Calbiochem (San Diego, CA). eNOS kit was purchased from R&D Systems (Minneapolis, MN, USA).

Uptake assays were performed as described previously 16. Briefly, cells were incubated in Krebs–Ringer phosphate HEPES (KRPH) buffer containing [³H]‐labeled substrate (1 μCi·mL⁻¹) at 37 °C for 2–60 min (for time‐dependent uptake) or 5 min (for dose‐dependent uptake and Na⁺/Cl⁻/H⁺‐dependent uptake). [³H]‐labeled noradrenaline, dopamine, serotonin, and histamine were purchased from PerkinElmer (Waltham, MA, USA) and [³H]‐1‐methyl‐4‐phenylpyridium acetate (MPP⁺) was purchased from American Radiolabelled Chemicals (St. Louis, MO, USA). Specific uptake rates in transfected cells were calculated by subtracting uptake in mock‐transfected CHO‐K1 cells.
The dependence of transporter activity on extracellular Na⁺ and/or Cl⁻ was examined by comparing MPP⁺ transport in KRPH buffer, Na⁺‐free buffer, and Cl⁻‐free buffer 10. The influence of extracellular pH on transport activity was investigated by comparing transport in pH 6.6 MES buffer, pH 7.4 HEPES buffer, and pH 8.2 HEPES buffer. Drug inhibition assays were performed using decynium‐22, imipramine hydrochloride, tetraethylammonium (TEA) chloride, cimetidine (Sigma‐Aldrich, St Louis, MO, USA), and corticosterone (Wako). The effects of protein kinase inhibitors on mOct3 activity were investigated using H‐89, RO‐32‐0432, and KN‐93 (all from Sigma‐Aldrich).

EGFR inhibitor PD 153035, pertussis toxin (PT), wortmannin (WT), PD98059 (PD), SB202190 (SB), and Ro-32-0432 (RO) were obtained from Sigma and were used on hHSC at concentration previously described20 (link).