Figure 4.
Enzymatic activity assays with B4GALT1 and ST6GAL1. A, lectin staining of COS-7 cells expressing WT and mutant B4GALT1 and ST6GAL1. Pictures of stained cells were taken using OperettaTM (PerkinElmer Life Sciences) using blue, green, and red filters. Blue represents nuclear Hoechst stain, green represents bound SNA-fluorescein on α-2,6 sialic acid, and red-orange represents bound RCA-I–rhodamine on β-1,4 galactose. Scale bar = 200 μm. B, RCA-I–rhodamine mean intensities as percent of up to 50,000 Lec20 cells expressing the depicted constructs (mean ± S.D., n = 3). Statistically significant changes relative to control cells (mock-transfected cells) are indicated (***, p < 0.001). C, SNA-fluorescein mean intensities as percent of up to 50,000 Lec 20 cells expressing the depicted constructs (mean ± S.D., n = 3). Statistically significant changes relative to control cells (mock-transfected cells) are indicated (**, p < 0.01; ***, p < 0.001). D, enzymatic activity of the ST6Gal mutants. Cells transfected with the indicated ST6GAL1 constructs were lysed in radioimmune precipitation assay buffer 24 h post-transfection, and then their activities were determined as described under “Experimental procedures” using asialofetuin as an acceptor. The values shown (columns) are expressed as the mean percent of disintegrations per minute (n = 3) after normalizing them against the ST6GAL1 protein present (determined by immunoblotting). Statistically significant changes relative to control cells (mock-transfected cells) are indicated (*, p < 0.05; ***, p < 0.001).