Fig. 2

Detection of polySia in cell lysates and cell culture supernatants; mRNA levels of polysialyltransferases. a PolySia was visualized in cell lysates using mAb 735. For negative control, sample was treated with endoN. b PolySia was enriched from cell culture supernatants with the help of mAb 735-coupled beads, and subsequently analyzed by Western blotting using antibody against polySia. c After immunoprecipitation of polySia from cell lysate (CL) and the corresponding cell culture supernatant (CM), polySia was visualized by Western blotting. d, e Total RNA was isolated from HUVEC and reverse-transcribed to cDNA. d Semi-quantitative PCR was performed to visualize the abundance of ST8SiaII and ST8SiaIV transcripts. GAPDH was used as reference. One representative gel-electrophoresis picture is shown. e Fluorescence-based quantitative RT-PCR was used to determine the number of ST8SiaII and ST8SiaIV transcripts in HUVEC (normalized against GAPDH, ACTB and EEF1A1 and calculated to 1 µg of total RNA, which was used as template for cDNA synthesis). Error bars indicate the standard deviation (n = 6)