Anti pkc

All drugs used in these experiments were the highest purity available commercially. L-NAME, indomethacin, acetylcholine, chelerythrine, rauwolscine, and ML-7 hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 was obtained from Calbiochem (La Jolla, CA, USA). Anti-PKC, anti-phospho-PKC (Pan), anti-MLC₂₀, and anti-phospho-MLC₂₀ at Ser19 were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-CPI-17 and anti-phospho-CPI-17 at Thr38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fura-2/AM was obtained from Molecular Probes (Eugene, OR, USA). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, streptomycin, trypsin/EDTA, and glutamine were supplied by Gibco BRL (Rockville, MD, USA). All concentrations are expressed as the final molar concentration in the organ bath. indomethacin and ML-7 hydrochloride were dissolved in dimethyl sulfoxide (final organ bath concentration, <0.1%). Unless otherwise stated, all drugs were dissolved and diluted in distilled water.

Total proteins collected from the transverse colons of subset groups (No, GEGR, Lop+vehicle, Lop+LoGEGR, Lop+MeGEGR, Lop+HiGEGR and Lop+BS treated SD rats) were separated by 4%–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 3 h, after which the resolved proteins were transferred to nitrocellulose membranes for 2 h at 40 V. Each membrane was then incubated separately with primary antibody, anti-mAChR M2 antibody (Alomone Labs, Jerusalem, Israel), anti-PI-3K (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-p-PI3K (Cell Signaling Technology Inc., Cambridge, MA, USA), anti-mAChR M3 antibody (Alomone Labs, Jerusalem, Israel), anti-PKC (Cell Signaling Technology Inc.), anti-p-PKC (Cell Signaling Technology Inc.), anti-Gα (Abcame, Cambridge, UK) or anti-actin (Sigma-Aldrich Co.) overnight at 4°C. Next, the membranes were washed with washing buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, and 0.05% Tween 20) and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Zymed Laboratories, South San Francisco, CA, USA) at a dilution of 1:1,000 and room temperature for 2 h. Finally, the membrane blots were developed using Chemiluminescence Reagent Plus kits (Pfizer, New York, NY, USA and Pharmacia, New York, NY, USA).

We used pharmaceuticals and chemicals that met the highest purity standards and were readily available in the market. Dexmedetomidine, NaF, GF109203X, L-NAME, and PDBu were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-PKC, anti-phospho-PKC, anti-MLC₂₀, and anti-phopho-MLC₂₀ antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Y-27632 was provided by Calbiochem (La Jolla, CA, USA). Anti-ROCK-2 antibody was purchased from Santa Cruz (CA, USA). PDBu and GF109203X were dissolved in dimethyl sulfoxide, with the final concentration of dimethyl sulfoxide being <0.1%. Unless specified otherwise, all medications were dissolved and thinned with distilled water.

Whole protein from NRCMs or mouse heart tissue was extracted as previously reported 11 (link). Intercellular signaling was analyzed using a Pathscan Intercellular Signaling Array kit (#7323 and #12856; Cell Signaling Technology, Inc.) following the protocol provided, and routine western blotting procedures were performed according to our previous report 11 (link). The primary antibodies used were as follows: anti-ANP (mouse mAb; 1:300; sc-515701; Santa Cruz Biotechnology, Inc.), anti-BNP (rabbit pAb; 1:500; ab19645; Abcam), anti-MYH7 (mouse mAb; 1:300; sc-53089; Santa Cruz Biotechnology, Inc.), anti-Erk1/2 (rabbit mAb; 1:1000; #9102), anti-phospho-Erk1/2 (Thr202/Thr204) (rabbit mAb; 1:1000; #9102), anti-Akt (rabbit mAb; 1:1000; #9272), anti-phospho-Akt (Ser473) (rabbit mAb; 1:1000; #4060), anti-PKC (rabbit mAb; 1:1000; #2056), anti-phospho-PKC pan (Thr514) (rabbit mAb; 1:1000; #9379), anti-AMPK (rabbit mAb; 1:1000; #5831), anti-phospho-AMPK (Thr172) (rabbit mAb; 1:1000; #2535), and anti-GAPDH (rabbit mAb; 1:2000; #2118;Cell Signaling Technology, Inc.). The secondary antibodies used were horseradish peroxidase (HRP) conjugated (GE Healthcare Life Sciences, Beijing, China): anti-mouse IgG, HRP-conjugated whole Ab sheep (NA931) or anti-rabbit IgG, HRP-linked whole Ab donkey (NA934).