Quantitative Binding Assay for IGFBP-3 and CD44

Most of the material used was purchased as we previously reported [48, 60, 66]. Phosphate‐buffered saline (PBS), streptavidin‐conjugated horseradish peroxidase (HRP), phenylmethylsulfonyl fluoride (PMSF), TCEP‐HCl, N‐ethylmaleimide, and HA‐biotin (B1557) were purchased from Sigma‐Aldrich (Burlington, MA, USA). PNGase F was from New England Biolabs (Ipswich, MA, USA). Recombinant human IGFBP‐3 protein (YCP1009, UniProt accession ID: P17936) was purchased from Speed BioSystems (Gaithersburg, MD, USA). Human recombinant CD44 protein (12211‐H08H) was purchased from Sino Biological (Wayne, PA, USA). Nonglycosylated recombinant IGFBP‐3 (MBS142177) was from MyBioSource (San Diego, CA, USA). HN (018‐26) and biotin‐HN (B‐018‐26, UniProt accession ID: Q8IVG9) were purchased from Phoenix Pharmaceuticals (Burlingame, CA, USA). CD44 antibody (5F12) (MA5‐12394), mouse IgG isotype control (mIgG), ultra 3,3′,5,5′‐tetramethylbenzidine (TMB)‐ELISA substrate solution, Halt Protease and Phosphatase Inhibitor Cocktail, and Nunc MaxiSorpTM 96‐well flat‐bottom plates were from Thermo Fisher. The SuperSignal West Pico Luminol (Chemiluminescence) Reagent and BCA Protein Assay Kit were from Pierce (Dallas, TX, USA).