Journal: The Journal of Biological Chemistry
Article Title: β-Galactoside α2,6-Sialyltranferase 1 Promotes Transforming Growth Factor-β-mediated Epithelial-Mesenchymal Transition *
Figure Lengend Snippet: St6gal1 is specifically up-regulated at the transcriptional level during the TGF-β-induced EMT in GE11 cells. A , RT-PCR using total RNA extracted from TGF-β-treated and -untreated cells was carried out to examine the expression levels of genes involved in protein sialylation. The expression level of Gapdh was used as a loading control. Sct , sialic acid transporter; St3gal , β-galactoside α2,3-sialyltransferase; ST6Gal , β-galactoside α2,6-sialyltransferase; St6galnac , α- N -acetylgalactosaminide α2,6-sialyltransferase; Neu , neuraminidase. B , cells treated with or without TGF-β were incubated with ( heavy line ) or without ( gray shadow ) biotin-conjugated MAA (recognizing α2,3-sialylated proteins) or biotin-conjugated SNA (recognizing α2,6-sialylated proteins), followed by incubation with streptavidin Alexa Fluor 647 conjugate, and subjected to FACS analysis. C , representative glycan profiling in the cells treated with or without TGF-β to compare the major complex type of N -glycans by LC/MS. Peak area of asialo- and sialylated N -glycans were calculated based on the extracted mass chromatograms acquired in positive and negative modes, respectively. The relative peak area of major N -glycans from those cells was presented as a percentage of the total peak area of the glycans. Glycan structures were deduced from the accurate masses and product ion spectra.
Article Snippet: Then cells were stained with the 10 μg/ml biotinylated Sambucus nigra lectin (SNA), which preferentially recognizes the α2,6-sialylated products or Maackia amurensis agglutinin (MAA), which preferentially recognized α2,3-sialylated products for 30 min on ice, followed by incubation with streptavidin-conjugate Alexa Fluor 647 (Invitrogen) for 30 min on ice.
Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, FACS, Liquid Chromatography with Mass Spectroscopy