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. 2012 Mar 1;287(17):13620–13632. doi: 10.1074/jbc.M111.304857

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — Increased expression of HAS2 is associated with increased production of HA in fro/fro (NSMase2^−/−) fibroblasts. A, the amount of hyaluronan (HA) in 7-day conditional culture medium from fro/fro (NSMase2^−/−) fibroblasts was >15-fold higher than that in WT culture media. HA levels were determined with a competitive ELISA kit from Echelon Biosciences (catalog number K-1200) according to the manufacturer's instructions. B, RT-PCR showed significantly increased expression of Has2 but not Has1 and Has3 in fro/fro (NSMase2^−/−) fibroblasts. C, Western blot showed increased HAS2 protein in fro/fro (NSMase2^−/−) fibroblasts compared with WT. Left panel, Western blot; right panel, quantification corrected for protein. D, increased HAS2 immunostaining of fro/fro (NSMase2^−/−) fibroblasts compared with WT. E, transfection of the fro/fro (NSMase2^−/−) fibroblasts with Smpd3 reversed the increased HAS2 expression. Left panel, Western blot; right panel, quantification corrected for protein. F, transfection of the fro/fro (NSMase2^−/−) fibroblasts with Smpd3 also reversed the increased HA synthesis. G, increased HAS2 expression is associated with lipid rafts in the fro/fro (NSMase2^−/−) fibroblasts. Western blot analysis using anti-flotillin and anti-HAS2 antibodies (as described in the text) showed the association of HAS2 and flotillin (lipid rafts fractions 3/4 marker), and increased lipid rafts HAS2 in the fro/fro (NSMase2^−/−) fibroblasts compared with WT. The procedures are as described in the text and results are representative of three independent experiments.