Arthrobacter ureafaciens neuraminidase

Peroxidase conjugated ABC reagent, Vectastain Elite ABC standard kit was from Vecter Laboratories, Inc. (Burlingame, CA, USA). Antihuman AGP rabbit serum and Protein Block Serum-Free Reagent were from Dako (Carpinteria, CA, USA). Peroxidase conjugated anti-human AGP and Universal HIER antigen retrieval reagent were from Abcam (Cambridge, UK). KPL SureBlue TMB Microwell Peroxidase Substrate was from Sera Care Life Sciences (Milford, MA, USA). Anti-PD-L1 and SignalStain Boost IHC Detection Reagent were obtained from Cell Signaling Technology (Danvers, MA, USA). N-Histofine High Stain HRP (Multi) was from Nichirei Biosciences Inc. (Tokyo, Japan). PNGase F was from Roche Applied Science (Indianapolis, USA). BlotGlyco was obtained from Sumitomo Bakelite, Co. (Tokyo, Japan). Neuraminidase (Arthrobacter ureafaciens, 1U/ml) was purchased from Nacalai Tesque (Kyoto, Japan). Biotinylated Aleuria aurantia lectin (AAL) was kindly provided by Prof. Naohisa Kochibe, Gunma University. Tumor-associated antigens in serum samples measured in this study were carcinoembryonic antigen (CEA) and squamous cell carcinoma (SCC). Levels of each antigen were determined by an ELISA using the Cobas system (Roche for CEA) and the ARCHITECT system (Abbot for SCC) and standard cut-off values were set at 5.0 ng/ml for CEA and 1.5 ng/ml for SCC, respectively.

Anti-TRA-1-60 (clone TRA-1-60, mouse IgM) was obtained from R&D Systems Inc. (Minneapolis, MN, USA). Horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig was obtained from Agilent Technology (Santa Clara, CA, USA). Polyclonal goat anti-podocalyxin IgG and HRP-conjugated rabbit anti-goat IgG secondary antibodies were obtained from R&D Systems. Anti-human iPSC/ESC, R-10G (mouse IgG1), and R-17F (mouse IgG1) antibodies were prepared as described previously [6 (link),8 (link)].
Peptide N-glycanase (PNGase F; recombinant protein from Escherichia coli) was obtained from Roche Diagnostics GmbH (Mannheim, Germany), neuraminidase (Arthrobacter ureafaciens) from Nacalai Tesque (Kyoto, Japan), α1-3/4 fucosidase from TaKaRa Bio, Inc. (Shiga, Japan), and α1-2 fucosidase from New England Biolabs (Ipswich, MA, USA). Chondroitinase ABC (Proteus vulgaris), heparinase mixture (heparinase, heparitinase I, and heparitinase II), keratanase (Pseudomonas sp.), keratanase II (Bacillus sp.), and endo-β-galactosidase (Escherichia freundii) were obtained from Seikagaku Biobusiness (Tokyo, Japan).