Figure 1

Biochemical analysis of GCTM-5 antigen complex. (A) Native PAGE immunoblot of SW1990 cell line supernatant with GCTM5. Position of 720 kDa marker is indicated. (B) Native PAGE immunoblots of SW1990 cell line supernatant (left hand track in each panel) and purified GCTM5 antigen complex (right hand track in each panel) probed with either anti-GCTM5 or CA19-9. Position of 720 kDa marker is indicated. (C) Immunoblot of immunoprecipitated GCTM5 antigen complex from SW1990, CFPAC-1, BXPC-3 and Capan-2 cell line supernatants separated on SDS-PAGE reducing gel, and probed with GCTM-5 or CA19-9. Position of 100 kDa marker is indicated. (D) Immunoblots of SW1990 cell line supernatant (lane 1) or purified GCTM-5 antigen complex (lane 2) separated on SDS-PAGE reducing gel and probed with GCTM-5, or antibodies to Mucin 16, Mucin 2, Mucin 1 and Mucin 5 A. Position of 100 kDa marker is indicated. (E) Native PAGE immunoblot of SW1990 cell line supernatant probed with ENPRO1. Position of 720 kDa marker is indicated. (F) Immunoblot of GCTM-5 immunoprecipitated antigen complexes from SW1990, CFPAC-1, Panc 10.05, BXPC-3 and Capan-2 cell line supernatants separated on SDS-PAGE reducing gels and probed with ENPRO1. Position of 100 kDa marker is indicated. (G) Immunoblot of immunoprecipitated SW1990 cell line GCTM-5 antigen complex separated on SDS-PAGE reducing gels and probed with anti- LGALS3BP. Positon of 100 KDa marker is indicated. (H) Immunoblot of SW1990 cell line supernatant (left hand track) and anti-LGALS3BP immunoprecipitate (right hand track) separated on SDS-PAGE reducing gel and probed with GCTM-5. Position of 100 kDa marker is indicated. (I) Immunoblot of SW1990 and CFPAC-1 cell line supernatants (left hand track) or extracellular vesicle preparations (right hand track) probed with GCTM-5. (J) Serial confocal microscope z-sections of live cell images of SW1990 or HuCC-TI cells incubated with Lysotracker dye and GCTM-5.