Additional file 1: Figure S1.

Conformation of DGKθ expression patterns by in situ hybridization and western blotting. (A) High-magnification images showing the cardiac end of the stomach, and dorsal skin at E17.5. Epithelium (ep), dorsal skin (ds). (B) Sagittal sections showing the DGKθ mRNA expression by in situ hybridization analysis at E17.5. In situ hybridization were subjected to general method with digoxigenin (DIG)-labeled RNA antisense probe and visualized by anti-DIG antibody conjugated with alkaline phosphatase and the substrate NBT/BCIP (Roche). The specific probe (541–967 bp: the length 427 bp) were generated by DIG RNA Labeling kit [T7 RNA polymerase] using purified mouse DGKθ PCR product (541–967 bp: the length 427 bp) which produced by the following primers and mouse whole brain cDNA library : 5’-AGGGAGGGGAACCTGCCTTC-3’ and 5’-GATCGAATTCTAATACGACTCACTATAGGGCCTCATTCCGAGCCAGGCGGG-3’. The deparaffinized sections were incubated for 16 hr at 42°C in 4 × SSC containing 40% formamide, 0.1 × Denhardt’s solution, 10 mM DTT, and DIG-labeled RNA antisense probe (1 μg/ml). After hybridization, the sections were washed twice for 15 min at 37°C with 2 × SSC and subsequently washed twice for 5 min at 37°C with 0.4 × SSC. (C) Estimation of the specificity of the anti-DGKθ antibodies. Adult mouse heart and whole brain were lysed in RIPA buffer containing protease inhibitor cocktails (Nacalai tesque). Total cell lysate (30 μg protein) was applied to SDS-polyacrylamide gel electrophoresis (7.5%) and subjected to western blotting with anti-DGKθ antibody diluted (antibody #1, 1:100; antibody #2, 1:200) in Can Get Signal solution (Toyobo). The numbers on the right margin represent the molecular sizes of the pre-stained protein standard.

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Ueda et al. BMC Developmental Biology 2013 13:35   doi:10.1186/1471-213X-13-35