50 confluent cultures of Caco-2 cells transiently transfected with the pN3XFlag-hTRPV

AZD0156 site Author manuscript; available in PMC 2013 October 01.Cui et al.PageWestern blot analysis of whole cell extracts from mucosal scrapings from different segments of wild type (WT) and transgenic mice (TG).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Bone Miner Res. Author manuscript; available in PMC 2013 October 01.Cui et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFigure 3. Calcium (Ca) absorption, serum Ca, and skeletal phenotypes in wild type (WT) and TRPV6 transgenic (TG) miceNIH-PA Author Manuscript8-wk-old WT and TG mice were fed a 0.25 Ca diet from weaning.50 confluent cultures of Caco-2 cells transiently transfected with the pN3XFlag-hTRPV6. Two days after transfection cell surface proteinsJ Bone Miner Res. Author manuscript; available in PMC 2013 October 01.Cui et al.Pagewere labeled by biotinylation and biotin-labeled proteins were isolated using an Avidinbound agarose column. A 12.4 kb villin promoter/enhancer was used to drive expression of a 3XFlag-tagged-human TRPV6 cDNA transgene in the intestine epithelial cells. Primers P1 and P2 were used for genotyping; primers P3 and P2 were used for assessing transgene mRNA levels. (B) PCR analysis of transgene mRNA expression in mouse tissues (Dd, duodenum; Je, jejunum; IL, ileum; CoP, proximal colon; CoD, distal colon). (C) Total TRPV6 mRNA levels (mouse and transgenic) in the intestinal segments of wild type and transgenic mice (TG) was determined by real-time PCR as described in methods. Bars represent mean ?SEM, n=4. (D) TRPV6 and -actin protein levels were determined byJ Bone Miner Res. Author manuscript; available in PMC 2013 October 01.Cui et al.PageWestern blot analysis of whole cell extracts from mucosal scrapings from different segments of wild type (WT) and transgenic mice (TG).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Bone Miner Res. Author manuscript; available in PMC 2013 October 01.Cui et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFigure 3. Calcium (Ca) absorption, serum Ca, and skeletal phenotypes in wild type (WT) and TRPV6 transgenic (TG) miceNIH-PA Author Manuscript8-wk-old WT and TG mice were fed a 0.25 Ca diet from weaning. (A) Serum Ca. (B) Duodenal Ca absorption. (C) fpsyg.2017.00007 Femoral bone mineral density (BMD) was determined using dual energy x-ray absorption (DEXA). Author manuscript; available in PMC 2013 October 01.Cui et al.Pagewere labeled by biotinylation and biotin-labeled proteins were isolated using an Avidinbound agarose column. Whole cell extracts (WCE), flow-through from the column (unbound), and material bound to the column (bound) from non-labeled cells (N) and biotinlabeled cells (B) were examined for 3XFlag-tagged hTRPV6 by Western blot analysis.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Bone Miner Res. Author manuscript; available in PMC 2013 October 01.Cui et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFigure 2. Generation of intestine-specific, flag-tagged human TRPV6 expressing transgenic mice(A) Transgene construct. A 12.4 kb villin promoter/enhancer was used to drive expression of a 3XFlag-tagged-human TRPV6 cDNA transgene in the intestine epithelial cells. Primers P1 and P2 were used for genotyping; primers P3 and P2 were used for assessing transgene mRNA levels.