Fig. 5. Analysis of STxB sulfation in SNX1- and SNX2-suppressed cells. The levels of TGN-localized STxB in control and SNX1- and SNX2-suppressed cells (individually and jointly) was quantified using a biochemical sulfation assay (see Materials and Methods and text for details). (A) Representative western blot showing levels of siRNA-mediated protein suppression. The table summarizes quantified levels of suppression achieved (ImageQuant) from n4 assays (n=6, for SNX1 and SNX2 individually). (B) The autoradiograph shows a typical result of a sulfation experiment using the B-(Sulf₂) construct. The graph summarizes results from six independent sets (each in duplicates) as percent of control. Autoradiographs were quantified by densitometry and normalized to overall levels of protein sulfation. In SNX1-suppressed cells, sulfation was reduced by 43±24.4% compared with control cells, whereas in SNX2-suppressed cells sulfation was reduced by only 22±26.7%; values are given as the mean ± standard deviation (s.d.). (C) Levels of siRNA-mediated suppression as determined by western blotting and quantified by densitometry were plotted against the levels of STxB sulfation (each data point represents mean from duplicates, normalized to global sulfation) for six experimental sets (pink triangles, SNX1 siRNA; blue triangles, SNX2 siRNA). Lower levels of SNX1-suppression (<90%) do not result in reduced sulfation (asterisks). The regions were determined using the lowest suppression level for SNX1 (x-axis) not inducing a reduction in STxB sulfation (100%, y-axis). (D) Graph shows mean levels (± s.d.) of overall protein sulfation for the different siRNA conditions from at least five different experiments (in duplicates).