universal sulfotransferase activity kit  (R&D Systems)

Journal: The Journal of Biological Chemistry

Article Title: Arylsulfatase B induces melanoma apoptosis by the ubiquitin ligase COP1

doi: 10.1016/j.jbc.2025.110402

Figure Lengend Snippet: Impact of rhARSB on sulfated glycosaminoglycans, chondroitin sulfotransferases, and chondroitin disaccharides . A , in the B16F10 lung melanomas, total sulfated glycosaminoglycans (sGAG) and chondroitin 4-sulfate (C4S) were measured by the dimethylmethylene blue assay and for C4S following immunoprecipitation by specific C4S antibody. They were significantly reduced by rhARSB treatment. B , chondroitin 4-sulfate (C4S) fluorescent immunostaining of ARSB-treated and control pulmonary melanoma tissue confirms the marked decline in C4S following rhARSB treatment. Bar = 150 μm. DAPI-stained nuclei ( blue ) and green fluorescent C4S are compared. C , in the A375 cells, C4S content was reduced by rhARSB and increased when ARSB was silenced. D , total sulfotransferase activity declined in the pulmonary melanoma tissue following rhARSB treatment. E , mRNA expression of CHST (carbohydrate sulfotransferase) 15 was reduced in the lung melanomas following exogenous ARSB. In contrast, the CHST11 expression was significantly increased. F . representative images of CHST15 immunofluorescence confirm decline in CHST15 protein in the ARSB-treated lung tissue, compared to the untreated control. Bar = 150 μm. DAPI-stained nuclei are blue and FITC-stained CHST15 are green . G, in A375 cells, CHST11 expression is inhibited by SIS3, a specific inhibitor of Smad3. ARSB silencing reduces CHST11 expression, and rhARSB increases CHST11 expression. H , in contrast, CHST15 mRNA expression is not inhibited by SIS3, but is inhibited by mithramycin (MM). This is consistent with a transcriptional mechanism requiring galectin-3 and Sp1 (Supplementary Information 5 and References 23 and 25). I and J , disaccharide analysis shows marked increase in 4S disaccharides following ARSB silencing, in contrast to decline following treatment by rhARSB. This suggests that the hydrolysis of the 4-sulfate group by rhARSB exceeds the capacity of CHST11 to increase the C4S content. ARSB knockdown increases the 4-sulfate disaccharide unit to over 22 μg/mg protein. 4S6S disaccharides, as present in chondroitin sulfate E and synthesized by CHST15, increase following ARSB knockdown, but are present at a concentration of <1 μg/mg protein.

Article Snippet: Total sulfotransferase activity was determined using the Universal Sulfotransferase Activity kit (R&D Systems) which detects activity of all sulfotransferases present.

Techniques: Dimethylmethylene Blue Assay, Immunoprecipitation, Immunostaining, Control, Staining, Activity Assay, Expressing, Immunofluorescence, Knockdown, Synthesized, Concentration Assay