Fig. 2.

T47D cells contains more membrane bound sialic acids as compared to HB4A, would be good model to compare as there are striking differences between the lectin binding characteristics of the different cell types. Cells were treated in the presence or absence of 10 mM Neu5Ac for 2 hours under nutrient deprivation, lysed by hypotonic shock in 1 ml water (15 min, 4 °C) and surface Neu5Ac content was determined by HPLC analysis. (A) Bar diagram showing quantitation of surface Neu5Ac. (B) Detection of ST3Gal-I and ST6Gal-I activities. Cells were lysed and the same amounts of proteins were used on solid phase assays. The α-2,3 and α-2,6 sialylated glycans of resultant fetuin was detected with biotinylated MAL-I and biotinylated SNA, respectively. (C) Quantitative RT-PCR of ST3Gal-I and ST6Gal-I. Cells were treated in the presence or absence of 10 mM Neu5Ac for 2 hours under nutrient deprivation. The relative mRNA expression of ST3Gal-I and ST6Gal-I was assessed by quantitative RT-PCR and the results were normalized to β-actin expression. The data are representative of four independent experiments with S.D. indicated by error bars. P values are determined by two-tailed student t-test and <0.05, <0.01 and <0.001 are indicated by one, two and three asterisks, respectively.