Fig. 6
PolySia cell surface expression is inhibited in TE671 rhabdomyosarcoma cells expressing α ST8 Sia II and α ST8 Sia IV intrabody. a Proliferation assay of 5 x 104 TE671 cells stable expressing αST8SiaII-IB and αST8SiaIV-IB, anti-NCAM IB or empty vector cultured in wells of a 6-well microtiter plate over a time period of 8 days. The number of living cells was determined after staining with trypan blue using a Rosenthal-Fuchs counting chamber. Starting material was 5 × 104 cells. The proliferation assay was done 3 times, bars demonstrate standard deviation calculated from the mean values. b Immunofluorescence analysis of anti-ST8SiaII-IB, anti-ST8SiaIV-IB and anti-NCAM intrabody expression. 106 recombinant CHO cells transiently transfected for 48 h with the anti-PolySTs intrabodies or anti-NCAM intrabody in a 6-well microtiter plate were analyzed by immunofluorescence. Anti-ST8SiaII, Anti-ST8SiaIV-IB and anti-NCAM intrabody were detected in red. c Flow cytometric detection of polySia and NCAM cell surface expression. Stable transfected cell lines are shown with red lines. Negative controls are non-transfected cells (grey line) and cells only incubated with the secondary antibody (grey area). In the case of detection of polySia negative controls are cells treated with endosialidase (grey area). 104 cells in 300 μl PBS containing 2% FCS and 10 μg/ml propidiumiodide were measured. The flow cytometric analysis was performed 4 times. Shown is characteristic cell surface staining of NCAM and polySia from one experiment