30 minutes post injection, embryos were transferred to 0. 1X MMR and allowed to develop until stage 21 for in situ hybridization or stage 45 for organ placement score. www.selleckchem.com/products/Calcitriol-(Rocaltrol).html All experimental procedures involving the use of animals for experimental purposes were approved by the Institutional Animal Care and Use Committees and Tufts University Department of Lab Animal Medicine under the protocol number M2008 08. Scoring for Organ situs At stage 45, embryos were anesthetized with 5% tricaine and analyzed for position of 3 organs the heart, stomach, and gallbladder. Heterotaxia was defined as reversal in position of one or more organs. Only embryos with normal dorsoanterior development were scored to avoid scoring instances of secondary randomization due to errors in the dorso ventral or antero posterior axial patterning, and only clear left or right sided organs were scored.

Percent heterotaxia was calculated as the number with hetero taxia divided by the number of total scorable embryos, i. e. embryos normal in all other ways. A c2 test was used for further statistical analysis. Whole amount in situ hybridization Embryos were collected at different stages of development and fixed in MEMFA for 3 hours at room temperature and used for in situ hybridization as described in Harland. Plasmids containing Xenopus Mad3 and Xenopus HDAC cDNA were purchased from Open Biosystems and cloned in pCS2. The plasmids were linearized and anti sense probes for in situ hybridization were generated in vitro using DIG labeling mix from Invitrogen.

Xenopus embryo drug treatment Batches of embryos were separated into experimental and control groups and exposed to 0. 1X MMR or 0. 1X MMR containing 100 mM Sodium Butyrate during different stages of development. The drug was washed out and the embryos were allowed to develop until stage 21 for Nr1 in situ or until stage 45 for organ placement score. Western Blotting and Coimmunoprecipitation assay For Western blottings embryos were collected at stage 7 and homogenized in lysis buffer. The lysate was centrifuged for 15 minutes at 4 C and the supernatant was collected and frozen. The extracted proteins were subjected to SDS PAGE and blotted onto a PVDF membrane. After blocking with 5% skim milk and 0. 1% Tween 20 in PBS, membrane filters were incubated with an anti Mad3 or anti acetyl H4 overnight at 4 C.

Cilengitide The membranes were washed in PBT and incubated with secondary HRP conjugated antibody. Immunosignals were visualized with chemoluminiscence. For Co IP assays, embryos were injected at the 1 cell stage with Mad3WT flag or Mad3 5mut flag constructs and collected at stage 7 when 10 embryos were homogenized in lysis buffer. A total of 100 ul of embryo lysate was incubated with 2 ug anti 5HT or rabbit IgG for 3 hours at 4 C fol lowed by incubation with proteinA agarose for 1 hour at the same temperature. The beads were collected by centrifugation and washed in lysis buffer.