Fig 1: The short isoform of ST6GalNAc1 functions as a sialyltransferase in PCa cells and induces expression of sTnA. FLAG-tagged long and short isoforms of ST6GalNacI were purified from Flp-In HEK293 cells by immunoprecipitation. Isolation was confirmed by western blot (A, upper panel). The purified proteins were then used directly in an in vitro sialylation assay (A, lower panel). Phosphate standard (R&D Systems EA002) was used as a positive control and to produce a standard curve. Despite missing the first 132 amino acids ST6GalNAc1-short is still able to function as a sialyltransferase in vitro. B. Detection of ST6GalNAc1 by real-time PCR and western blot in DU145 cells stably transfected with either empty vector (lane 1), full length FLAG-tagged ST6GalNAc1 (lane 2), or FLAG-tagged ST6GalNAc1-short (lane 3). Detection of bands at just above 55kDa and 69kDa by western blot confirm the successful creation of stable cell lines expressing both isoforms. C. Immunofluorescence detection of the sTn antigen using the B72.3 antibody in DU145 cells stably expressing either a control empty vector, full length ST6GalNAc1 or ST6GalNAc1-short. There is increased detection of sTn in cells transfected with both isoforms of ST6GalNAc1. Images are representative of 3 independent experimental repeats.