Fig. 4

Production and characterization of mAb specific to autocatalytic loop. (a) mAb3F3 specifically binds to the autocatalytic loop. ELISA of mAb3F3 binding to the BSA conjugated Mat-Loop, and not to BSA only. (b) Competition human (Mat-Loop) and mouse loop (Epi-Loop) peptides to mAb3F3 binding on ELISA, coated with BSA conjugated human loop peptide. SC, scrambled sequence did not compete. (c) Modeling of mAb3F3. Red: H-CDR1, Yellow: H-CDR2, Blue: H-CDR3, Green: L-CDR1, Purple: L-CDR2, Gray: L-CDR3. (d) Docking modeling of mAb3F3 to Mat-loop peptides (lowest energy, − 238). Cyan: loop peptide. (e) Amino acids interacting with mAb3F3 in Mat-loop peptide were indicated as cyan. White arrow indicates the activation cleavage site. (f-i) Immunoprecipitation and western blotting for testing specific interaction of mAb3F3 to native Prss14/ST14 in cells. (f) Whole lysates of HEK293T cells transfected with full-length human Prss14 (Mat) and vector (Vec) were reacted with mAb3F3. (g) Whole lysates of HEK293T cells transfected with EGFP-S805A and EGFP was immunoprecipitated with mAb3F3 (h) Immunoprecipitation of MCF7. (i) Immunoprecipitation of 4 T1. (j) Flow cytometry of human Prss14/ST14 expressing HEK293T cells. Mat-Loop and Epi-Sc were used for competition. (k) Flow cytometry with CHO-S cells transiently transfected with CD8 or full length Prss14/ST14