Goat anti rabbit igg peroxidase antibody

The protein content in each sample was determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL). Quantified each sample concentration to 60–80 μg and add 4 × SDS sample dye and then denatured sample for 10 min at 95°C. Proteins were fractionated on SDS-PAGE, transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK) and detected with antibodies against IDO (Thermo Scientific, Rockford, IL), the mammalian target of rapamycin (mTOR) (Cell Signaling, Danvers, MA), phosphor-mTOR (Cell Signaling), protein kinase B (AKT) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA), phosphor-AKT (Santa Cruz Biotechnology, Inc.), p70S6K (Cell Signaling), phosphor-p70S6K (Cell Signaling), microtubule associated protein 1 light chain 3 (LC3) (Novus Biologicals, Littleton, CO), Beclin (Novus) and β-actin (Sigma Aldrich). Rabbit anti-mouse IgG-peroxidase antibody (Sigma Aldrich) and goat anti-rabbit IgG-peroxidase antibody (Sigma Aldrich) were used as the secondary antibody and protein-antibody complexes were visualized by enhanced chemiluminescence system (Amersham) [56 (link)]. The signals were quantified with ImageJ software (rsbweb.nih.gov/ij) [57 (link)].

The Bicinchoninic Acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL) was used to determine the protein contents. SDS-PAGE was used to fractionate protein and the fractionated proteins were transferred to nitrocellulose membranes (Pall Life Science, Glen Cove, NY). The antibodies against PD-L1 (GeneTex, Inc. Irvine, CA), phosphorylation-AKT (Santa Cruz Biotechnology Inc, Santa Cruz, CA), AKT (Santa Cruz), phosphorylation- p70s6K (Cell Signaling, Danvers, MA), p70s6K (Cell Signaling), phosphorylation-mTOR (Cell Signaling), mTOR (Cell Signaling), caspase 3 (GeneTex) or β-actin (Sigma-Aldrich, St. Louis, MO) were used to detect targeted protein. Rabbit anti-mouse IgG-peroxidase antibody (Sigma Aldrich) and goat anti-rabbit IgG-peroxidase antibody (Sigma Aldrich) were used as secondary antibodies. Chemiluminescence system (T-Pro Biotechnology, New Taipei City, Taiwan) was used to observe the signals. ImageJ software was used to quantify the signals 9 (link).

HBMEC/ciβ cells were cultured on dishes as described above. Homogenates were prepared using methods described previously [7 (link)]. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto a polyvinyldene difluoride membrane. The membrane was blocked with 5% skim milk. The primary antibodies used were rabbit anti-glucocorticoid receptor (GR) polyclonal IgG (1,000-fold dilution, sc-1002, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-VE-cadherin polyclonal IgG (1,000-fold dilution, sc-28644, Santa Cruz Biotechnology), rabbit anti-claudin-5 polyclonal IgG (1,000-fold dilution, ab53765, Abcam, Cambridge, UK), or rabbit anti-occludin polyclonal IgG (1,000-fold dilution, 71–1500, Zymed Laboratories, San Francisco, CA, USA). The secondary antibody used was goat anti-rabbit IgG–peroxidase antibody (10,000-fold dilution, A9196, Sigma).

The protein content was determined using bicinchoninic acid (BCA) protein assay (Pierce Biotechnology, Rockford, IL, USA). About 60-80 µg of protein from the lysates were loaded and fractionated on SDS-PAGE, transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham, Little Chalfont, UK) and detected with antibodies against P-gp (GeneTex, Inc. Irvine, CA, USA), the mammalian target of rapamycin (mTOR) (Cell Signaling, Danvers, MA, USA), phosph-mTOR (Cell Signaling), protein kinase B (AKT) (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA), phosph-AKT (Santa Cruz Biotechnology, Inc.), p70s6K (Cell Signaling), phosph-p70s6K (Cell Signaling), caspase 3 (Cell Signaling), β-actin (Sigma Aldrich). Rabbit anti-mouse IgG-peroxidase antibody (Sigma Aldrich) and goat anti-rabbit IgG-peroxidase antibody (Sigma Aldrich) were used as the secondary antibody. The protein-antibody complexes were visualized using enhanced chemiluminescence system. The Western blotting signals were quantified with ImageJ software (rsbweb.nih.gov/ij/) 20 (link).

The ELISA binding assay was performed according to Decreux and Messiaen (2005 (link)) with modifications. Nunc Maxisorp flat-bottom plate wells (Invitrogen, CA, USA) were coated with commercial pectin from citrus peel (P9135; Sigma-Aldrich, USA; https://www.sigmaaldrich.com) or pectin extracted from C. plantagineum leaves (250 µg ml⁻¹, 100 µl well^–1) at 4 °C overnight and then wells were incubated with the following solutions for the indicated time: 100 µl of 3% (w/v) low fat dried milk in wash buffer (20 mM Tris–HCl, 150 mM NaCl, pH 8.0) for 2 h; 50 µl of purified His-tagged CpGLP1 protein in binding buffer (1% low fat dried milk, 20 mM Tris–HCl, 150 mM NaCl, 2 mM CaCl₂, pH 8.0) for 2 h; wash buffer (four times, briefly); 50 µl of anti-His tag antibody (1:10,000) (Jung et al. 2019 (link)) in incubation buffer (1% low fat dried milk, 20 mM Tris–HCl, 150 mM NaCl, pH 8.0) for 1 h; wash buffer (four times, briefly); 50 µl of goat anti-rabbit IgG peroxidase antibody (1:10,000) (Sigma, A9169) in incubation buffer for 1 h; wash buffer (six times, briefly). The bound recombinant CpGLP1 protein was visualized in the presence of the TMB (3,3′,5,5′-tetramethylbenzidine) substrate (Sigma, T2885). The absorbance was measured at 450 nm after color development in the dark and the reaction was stopped by adding 50 µl of 10% (v/v) phosphoric acid.