TI - Hsulf-1 expression in pancreatic cancer cell lines . AB - QRT-PCR analysis was carried out in 8 cultured pancreatic cancer cell lines . This analysis revealed relatively high expression of Hsulf-1 mRNA in Su-8686 and moderate expression in T3M4 , Colo-357 and BxPc-3 pancreatic cancer cell lines . In the other cell lines , Hsulf-1 expression was below the detection level ( Figure 3 A ) . Panc-1 pancreatic cancer cells were selected for Hsulf-1 transfection , since Hsulf-1 expression was below the level of detection in this cell line . To confirm successful transfection of Panc-1 cells with the full-length Hsulf-1 construct , Northern blot analysis was carried out using a Hsulf-1 antisense riboprobe . A total of number 36 clones were screened , of which 10 clones clearly expressed Hsulf-1 mRNA . Two Hsulf-1 positive clones ( sulf-26 and sulf-38 ) were selected for use in further experiments and compared to empty vector-transfected ( EV ) and non transfected wild type ( WT ) Panc-1 cells ( Figure 3 B ) . To confirm the expression of Hsulf-1 in the positive clones on the protein level , immunoblotting was performed . Since the Hsulf-1 expression plasmid contained a c-myc tag [10] , it was possible to detect the expression of the Hsulf-1-myc fusion protein in the selected clones by immunoblot analysis with an c-myc antibody ( Figure 3 C ) . To determine the activity of the expressed sulfatase , cellular extracts prepared from both control ( wild type and empty vector ) and transfected clones ( sulf-26 , sulf-38 ) were analyzed. 4-Methylumbelliferyl-sulfate , which represents a subSTRate for a variety of sulfatases , including cellular steroid sulfatases , was used as the SUBstrate for sulfatase activity . Upon transfection , sulfatase activity was most prominently increased in ( Figure 3 D ) . However , since this assay could not differentiate between different sulfatases , high sulfatase activity was also observed in the control cells . Therefore , to further confirm successful transfection and increased Hsulf-1 activity , immunofluorescence with the 10E4 anti-HSPG monoclonal antibody , which recognizes N-sulfated glucosamine -containing HSPGs , was carried out . Prominent staining of the cell membrane was observed in both wild type ( Figure 4A ) and empty vector Panc-1 cells ( Figure 4 C ) . In contrast , markedly diminished staining of the cell membrane was observed in the two Hsulf-1 expressing clones ( Figure 4 B , D ) , indicating that Hsulf-1 desulfates HSPGs at the cell surface .