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SuppFigs1-3.tif18542KSuppl. Fig. 1. Immunofluorescent staining of Twist protein in vitro. After culturing for 24 hr, the Twist protein (green) was expressed in the epithelial layer where the MES would have formed. Nuclei were counterstained red. Scale bar = 37.5 μm. Suppl. Fig. 2. siRNA transfection efficiency in organ culture. The siRNA transfection efficiency was monitored by using a fluorescent oligo (Block-iT, Invitrogen). The fluorescein-labeled oligo was transfected to the tissue at a concentration of 200 nM and viewed with the confocal microscope as whole mount after 24 hr. A filter (488 nm) was used for detecting the oligo. Multiple optical sections were merged into a projected image. Scale bar = 75 μm. Suppl. Fig. 3. Western blot of Twist protein expression after siRNA culture. Whole palatal shelves were lysed in the RIPA buffer (RadioImmunoPrecipitation Assay Buffer, Sigma) and used for Western blots. The concentrations of total protein in each sample were determined by BCA assay (Pierce). Ten micrograms total protein was loaded in each well on a 4–12% NuPage Bis-Tris gel (Invitrogen). Protein was transferred to PVDF membrane (Millipore). The membrane was incubated with polyclonal primary antibody against Twist (1:1,000, Santa Cruz). Secondary antibodies conjugated with alkaline phosphatase were used at 1:1,000, visualized with the chemiluminescence (Invitrogen), and captured with the Kodak Image station 440<INF>CF</INF> (Kodak Digital Science). A single band at MW about 28 kDa was detected. Positive control (HeLa nuclear extract) migrated at the same mw as experimental samples.

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