Fig. 3

MS identified Nucleolin, GRP-78, α-Enolase and Annexin A2 display O-GalNAc glycosylation. a left: Tn antigen is expressed on Nucleolin, GRP-78, α-Enolase and Annexin A2. Immunoprecipitation and western blot analysis were performed using VVL lectin for Tn antigen precipitation and Nucleolin, GRP-78, α-Enolase and Annexin A2 antibodies for detection of specific protein signals. a right: Protein expression levels of the identified proteins in total cell lysates of COSMC knockdown as well as control cells remained unchanged, except for Annexin 2, which showed a decrease in Panc-1 COSMC knockdown cells. b Vice versa, immunoprecipitation using specific antibodies and detection with VVL lectin was performed as well. c Immunocytochemistry of Panc-1 COSMC knockdown cells and control cells. Left: Aberrant O-glycans, including Tn antigen, were detected using VVL-FITC conjugated lectin (green). Middle left: Nucleolin was detected (red) using an anti-Nucleolin antibody (ab13541). Middle right: Nuclei were stained with DAPI (blue). Right: Overlay of Tn antigen and Nucleolin immunocytochemistry. d Nucleolin, GRP-78, Enolase and Annexin detection in the cell membrane protein fraction. Her2 antibody was used as marker for membrane fraction and GAPDH antibody was used as marker for cytoplasmic proteins