Chymostatin

Hippocampal cultures with 15 DIV (90 × 10³ cells/cm²) were washed twice with ice-cold PBS, and once more with PBS buffer supplemented with 1 mM dithiothreitol (DTT) and a cocktail of protease inhibitors [0.1 mM phenylmethylsulfonyl fluoride (PMSF) and CLAP (1 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml antipain, and 1 μg/ml pepstatin; Sigma)]. The cells were then lysed with RIPA (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 5 mM EGTA, 1% Triton, 0.5% DOC, and 0.1% SDS at a final pH 7.5) supplemented with 50 mM sodium fluoride (NaF), 1.5 mM sodium ortovanadate (Na₃VO₄), and the cocktail of protease inhibitors. After sonication and centrifugation at 16,100 × g for 10 min at 4°C, protein in the supernatants was quantified using the bicinchoninic acid (BCA) assay kit (Pierce). Samples were then denaturated with 2× concentrated denaturating buffer (125 mM Tris, pH 6.8, 100 mM glycine, 4% SDS, 200 mM DTT, 40% glycerol, 3 mM Na₃VO₄, and 0.01% bromophenol blue) for 5 min at 95°C, and proteins of interest were analyzed by Western blotting. Alternatively, extracts were performed in a lysis buffer supplemented with 50 U/ml of RNase inhibitor (SUPERaseIn, Ambion Applied Biosystems) and samples processed for RNA coimmunoprecipitation experiments.

Polyclonal antibodies against: (i) synthetic peptides corresponding to sorghum C₄-PEPC C-terminus [(Y) 942EDTLILTMKGIAAGMQNTG960] and dephosphorylated N-terminus [4ERHHSIDAQLRALAPGKVSEE24(YG)], C19-IgG and N24-IgG, respectively, were purchased from NEOSYSTEM S.A. (Strasbourg, France); and (ii) native C₄-PEPC from sorghum leaves (PEPC-IgG) achieved as described in Pacquit et al. (1995) (link).
Phospholipids with varying format and fatty acyl chain composition were all purchased from Avanti Polar Lipids, (Alabaster, AL, United States). Lipids in chloroform stock were dried with gaseous N₂, rehydrated in 0.1 M Tris–HCl buffer (pH 8) and sonicated prior to adding to the assays. Protease inhibitors used were: Chymostatin (cysteine protease, chymotrypsin and elastase inhibitor); phenylmethane sulfonyl fluoride (PMSF; serine protease inhibitor); Aprotinin (serine protease inhibitor); Bestatin (aminopeptidase inhibitor); Leupeptin (serine and cysteine protease inhibitor); E-64 (selective cysteine protease inhibitor, such as cathepsin B and L); and protease inhibitor cocktail (AEBSF; serine proteases inhibitor; Phenanthroline, metalloproteases inhibitor; Pepstatin A, acid proteases inhibitor, Leupeptine, Bestatin and E-64) from Sigma Aldrich code P9599 (St Louis, MO, United States).

Angiotensin II (Peptide Institute, Osaka, Japan), losartan (Wako Pure Chemical Industries, Osaka, Japan), PD123,319 (Sigma-Aldrich, St. Louis, MO, USA), angiotensin I (Peptide Institute, Osaka, Japan), captopril (Sigma-Aldrich, St. Louis, MO, USA), chymostatin (Sigma-Aldrich, St. Louis, MO, USA) and nickel(II) chloride hexahydrate (Sigma-Aldrich, St. Louis, MO, USA) were dissolved in water. Carvedilol (Tokyo Chemical Industry, Tokyo, Japan), xestospongin C (Wako Pure Chemical Industries, Osaka, Japan), SEA0400 (synthesized in our faculty according to the reported method [75 (link)]), 2-APB (Sigma-Aldrich, St. Louis, MO, USA) and ryanodine (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in dimethyl sulfoxide (DMSO). They were added to the organ bath to obtain the desired final concentration.

MCF‐10A, MCF‐7, and MDA‐MB‐231 breast cell lines obtained from the ATCC cultured and stored following ATCC protocol of authentication by short terminal repeat analysis. The cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing high glucose (4.5 g/L) (Gibco/Life Technologies , Carlsbad, CA, USA) and supplemented with 7% fetal bovine serum (Harlan Bioproducts for Science, Inc., Indianapolis, IN, USA). Insulin, verapamil, MG132, chymostatin, leupeptin, pepstatin A, metformin, rapamycin, and PP242 purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Following antibodies were obtained from indicated distributors: anti-annexin A2 (rabbit monoclonal, (CST D11G2, Danvers, MA), anti-annexin A2 (mouse monoclonal 610068, BD biosciences), anti-caveolin-1 (rabbit polyclonal Sc-894, Santa Cruz), anti-cavin-1 (anti-PTRF, rabbit polyclonal AP7421a, Abgent, San Diego, CA), anti-S100A10 (mouse monoclonal 4E7E10, CST), anti-phosphotyrosine (mouse monoclonal 4G10, Merck Millipore, Darmstadt, Germany), anti-eNOS (mouse monoclonal 610296, BD Biosciences), anti-p-Src (Y418, BS4176) and anti-p-Src (Y529, BS4729) (rabbit polyclonal, Bioworld Technology, St. Louis Park, MN), Donkey anti-rabbit IgG HRP and anti-mouse IgG HRP (Promega). OLA (Wako), EPA (Nu-Chek Prep, Elysian, MN) and DHA (Cayman Chemicals, Ann Arbor, MI) were obtained from indicate distributors. Aprotinin (Wako), α-2-antiplasmin (Haematologic Technologies Essex Junction VT), 4-(2-Aminomethyl) benzensulfonyl fluoride (AEBSF, nacalai tesque, Kyoto Japan), chymostatin (Sigma-Aldrich, St. Louis, MO) and Gö6976 (LC Laboratories, Woburn, MA) were obtained from indicated distributors. Other reagents not specified were obtained from Wako.