Cnbr sepharose 4b

A serum pool collected from TZ adult donors with high anti- CHO-rPvs48/45 antibody titers was used for IgG purification, as described previously [72 (link), 73 (link)]. To prepare the antigen-Sepharose conjugate, CNBr-sepharose 4B (Amersham Bioscience AB, Uppsala, Sweden) was activated with 1 mM HCl. Then, 5 mg of the CHO-rPvs48/45 protein was dissolved in 1 mL of coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.0). The sera were diluted 5-fold with PBS (1x) containing 0.5 M sodium chloride and were mixed with the antigen-sepharose conjugate and stirred O/N gently at 4°C. The antigen-sepharose beads were then washed with 5 mL of TRIS (20 mM containing 0.5 M NaCl, pH 8.0) and then with 5 mL of TRIS (20 mM, pH 8.0). The bound antibody was eluted with a solution containing glycine (0.1 M, pH 2.5). The fractions (F1, F2, F3) were collected in TRIS solution (1 M, pH 8.0) to instantly neutralize the solutions before dialyzing them against phosphate buffer (0.1M, pH 7.0). The antibody (IgG) concentration of each fraction was determined by the absorbance of the solution at 280 nm [72 (link), 73 (link)]. In addition, ELISA was used to determine the recognition of rPvs48/45 by each of the purified antibody fractions.

6,7,4'-THIF was obtained from Chromadex™ (Irvine, CA, USA), and Dulbecco’s modified eagle medium (DMEM) and MMP-1 antibody were purchased from Thermo Fisher Scientific (San José, CA, USA). Medium 199 (M199) and daidzein were purchased from Sigma-Aldrich (St. Louis, MO, USA), and fetal bovine serum (FBS) was purchased from Gemini Bio-Products (Calabasas, CA, USA). CNBr-Sepharose 4B, [γ-32P]-ATP and the chemiluminescence detection kit were obtained from Amersham Pharmacia Biotech (Piscataway, NJ, USA). The protein assay kit was purchased from Bio-Rad Laboratories (Hercules, CA, USA). MTS solution was from Promega (Madison, WI, USA). Penicillin/streptomycin was purchased from Invitrogen (Grand Island, NY, USA). Primary antibodies recognizing phosphorylated MEK (Ser^217/221), total MEK, phosphorylated SEK1/MKK4 (MKK4, Ser²⁵⁷/Thr²⁶¹), phosphorylated MKK3 (Ser¹⁸⁹)/6 (Ser²⁰⁷), total MKK3, phosphorylated p38 (Tyr^180/182), total p38 and PKCα were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phosphorylated-ERKs (Tyr²⁰⁴), total ERKs, ERK2, total MKK4, phosphorylated JNK (Thr¹⁸³/Tyr¹⁸⁵), total JNK, JNK2 and phosphorylated PKCα (Ser⁶⁵⁷) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Active PKCα and PKCδ proteins were purchased from Millipore (Bedford, MA, USA).