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. Author manuscript; available in PMC: 2016 Mar 1.
Published in final edited form as: Biochim Biophys Acta. 2014 Dec 12;1849(3):342–352. doi: 10.1016/j.bbagrm.2014.12.009

Figure 3. BMP4 in culture medium media declined when ARSB was silenced, due to BMP4 sequestration by C4S in the cell membrane.

Figure 3

A BMP4 in the spent media of the NCM460 cells was markedly reduced following ARSB knockdown (p<0.001; one-way ANOVA with Tukey-Kramer post-test; n=6).

B. BMP4 was significantly reduced in the serum of the ARSB-null mice (p<0.01, unpaired t-test, two-tailed; n=5).

C. Following ARSB silencing, the membrane-bound BMP4 increased significantly (p<0.001, one-way ANOVA with Tukey-Kramer post-test; n=3). Treatment with chondroitinase ABC reduced the membrane-bound BMP4 (p<0.001, one-way ANOVA with Tukey-Kramer post-test; n=3). In contrast, there was no effect of keratanase treatment.

D. Representative Western blot demonstrates increase in the intestinal membrane-associated Bmp-4 of male and female ARSB-null mice, compared to gender and age-matched C57BL/6J controls.

E. The quantity of BMP4 that co-immunoprecipitated with C4S in the NCM460 cells increased significantly when ARSB was silenced by siRNA (p<0.0001, unpaired t-test, two-tailed; n=3 independent experiments).

F. Following treatment with chondroitinase ABC, the BMP4 in the spent media increased significantly when ARSB was silenced (p<0.001), although the cellular BMP4 and total BMP4 were reduced (p<0.001, p<0.01, respectively; one way ANOVA with Tukey-Kramer post-test; n=3).

G. mRNA expression of CHST 11 was unaffected by treatment with chondroitinase ABC (n=6). [ARSB=arylsulfatase B; BMP=bone morphogenetic protein; ChABC=chondroitinase ABC; F=female; CHST11=carbohydrate (chondroitin 4) sulfotransferase 11; Kase=keratanase; M=male; N.D.=no difference]