Rp 8 br pet cgmps

Expression, amplification, and purification of FIV envelope glycoprotein gp95 and capsid p26 recombinant proteins were performed using previously described methods [50 (link),101 (link)]. Briefly, gp95 was purified from Chinese hamster ovary (CHO) cells, and the human Fc tag, in frame with either protein, served as a means to purify the proteins using Staphylococcus Protein A-Sepharose [102 (link),103 (link)], or was isolated following transfection of HEK 293S cells with an expression vector. Gag antigen was expressed in Escherichia coli and purified using a GST-tag [101 (link)]. HIV CXCR4-tropic gp120 (IIIB) was obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 IIIB gp120 Recombinant Protein from ImmunoDX, LLC. HIV CCR5-tropic gp120 (JRFL) was obtained from Dr. C.A.L. Kassuya at the Federal University of Grande Dourados, Brazil [104 (link)]. The following inhibitors were used in this study: 200-nM AMD3100 (Tocris), 25-μM 2APB (Abcam), 10-μM Dantrolene (Abcam), 25-μM or 50-μM DL-APV (Abcam), 2-μM NPA (Tocris), 1-μM or 10-μM Rp8-Br-PET-cGMPS (Tocris) for cultured mouse or feline neurons, respectively, 10-μM or 30-μM CNQX (Abcam), 1-μM TTX (Tocris), 10-μM nifedipine (Abcam), and 20-nM human SDF-1 (Abcam).

The preparation of methanolic H. stoechas extract was previously described [12 (link)], and a plant voucher was deposited at the Universidad San Jorge herbarium (ref. 002–2014). The phenolic profile of this extract was previously determined [12 (link)] (see Introduction).
The compositions of the buffers used were as follows: Krebs buffer (in millimolar): NaCl 120, KCl 4.7, CaCl₂ 2.4, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, and glucose 5.6. Ca²⁺-free Krebs: NaCl 120, KCl 4.7, CaCl₂ 2.40, MgSO₄ 1.2, NaHCO₃ 24.5, KH₂PO₄ 1, glucose 5.6, and ethyleneglycoltetraacetic acid (EGTA) 1. Ca²⁺-free high K⁺ Krebs ([K⁺]_o = 50 mM). All buffers were adjusted to pH 7.4. Apamin (AP), arzanol, barium chloride dihydrate (BaCl₂), glibenclamide (Glib), heparin (H), H-89 dihydrochloride hydrate, indomethacine, Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME), phenylephrine (PE), ruthenium red (RR) and verapamil (V) were obtained from Sigma (Madrid, Spain).
Tetraethylammonium (TEA), 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and Rp-8-Br-PET-cGMPS were purchased from Tocris (Madrid, Spain). AP was diluted in acetic acid. Glib, TRAM-34 and arzanol were dissolved in dimethyl sulfoxide (DMSO). All other chemicals were dissolved in distilled water.