Fig. 1. Full-length ITIH5 (ITI) is secreted extracellularly and cleaved in PDAC cells, whereas secretion-deficient ITIH5Δs (Δs) is not secreted extracellularly.

a Organisation of ITIH5 domain structure labelled by amino acid (AA) position for ITI and Δs expression constructs expressed in the highly metastatic and low endogenous ITIH5-expressing S2-007 human PDAC cell line. Domain map adapted from Himmelfarb et al.³ Key features: cleavage site (CS); domains (grey): secretion signal peptide (SP), vault protein inter-α-trypsin domain (VIT), von Willebrand type A domain (vWA), multicopper oxidase domain (MCOD) and FLAG epitope (black). Labelled locations of ITIH5 and FLAG antibody binding. b Expression of full-length (ITI) and secretion-deficient ITIH5 (Δs) in S2-007 cells and immunoprecipitation (IP) from conditioned media to detect secreted ITIH5. ITIH5 antibody (binds on N-terminal side of cleavage site at AA578-606) and FLAG antibody (binds on the C-terminal side of CS after the end of ITIH5 open-reading frame). c Western blotting of extracellular vesicle (EV) preparations (particle-size range 20–300 nm, mean 155 nm measured using Nanosight^TM particle analysis device) for ITIH5. Only minimal ITIH5 is present in EVs. d, e Western blotting of ITIH5 in cellular fractions. Two separately conducted cellular fractionations are shown per blot where either cytoplasmic fraction was separated from nuclear fraction or membrane/membrane-associated fraction was separated from cytoplasmic fraction. Antibodies used were the N-terminal-detecting ITIH5 (D) or C-terminal-detecting FLAG (E). Extracellularly, large and small fragments of ITIH5 are present. f ITIH5 is detected in the cytoplasm and nucleus using immunocytochemistry. Scale bar is 25 µm. g Locations of predicted ITIH5 nuclear localisation signals as predicted by sequence recognition algorithm. The algorithm is described in detail.^22,23 Na/K, sodium/potassium ATPase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.