Anti mouse igg hrp conjugate

Aleuria aurantia lectin‐biotin and wheat germ hemagglutinin (WGA) were purchased from J‐OIL MILLS (Tokyo, Japan). Sambucus nigra lectin (SNA‐1) and its horseradish peroxidase (HRP) and biotin derivatives were obtained from EY Laboratories, Inc. (San Mateo, CA). Mouse monoclonal antibodies against sialyl‐Lewis^a (CA19‐9, NS19‐9) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Anti‐rabbit IgG‐HRP conjugate and anti‐mouse IgG‐HRP conjugate were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). BlotGlyco beads were obtained from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan).

The DNA fragments used in this study, listed in Supplementary Table S1, were chemically synthesized with an H8 DNA/RNA Synthesizer (K&A Laborgeraete), by using natural-base and biotin-dT phosphoramidites from Glen Research and a Ds phosphoramidite synthesized in-house, as described previously (42 (link)). The chemically synthesized DNAs were purified by denaturing gel electrophoresis. The mouse monoclonal anti-VEGF antibodies (clones 26503, VG76e, 16F1 and VG1) and anti-IFNγ antibodies (clones B133.5 and 2G1) were obtained from Thermo Fisher Scientific. Rabbit anti-biotin IgG (A150-109A) was purchased from Bethyl Laboratories, Inc. Recombinant human VEGF₁₆₅ and IFNγ were obtained from PeproTech. The streptavidin-HRP conjugate (1 mg/ml) and the anti-mouse IgG HRP conjugate (1 mg/ml) were obtained from Jackson ImmunoResearch and Promega, respectively. Stock solutions (10×) for PBS and D-PBS(–) were obtained from Thermo Fisher Scientific and Nacalai Tesque, respectively. BSA, streptavidin, and Tween 20 were purchased from Promega. Nonidet P-40 was purchased from Nacalai Tesque. The TMB-substrate solution was purchased from KPL. Human serum (lot # SLBS8635) was obtained from Sigma-Aldrich.

Western blotting was performed as described previously 14. Primary antibodies 1:1000 anti‐ZEB1 (Abcam), 1:1000 anti‐E‐cadherin (CST),1:1000 anti‐ZO1 (CST), 1:1000 anti‐N‐cadherin (CST), 1:1000 antivimentin (CST), and 1:10000 anti‐GAPDH (Santa Cruz) were used. Secondary detection antibodies were anti‐mouse IgG‐HRP conjugate (Jackson) used at 1:10000 or anti‐rabbit IgG–HRP conjugate (Jackson). Blots were developed with ECL substrate (Millipore Boston, MA) and analyzed on an imager (GE Healthcare, London, UK).