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DVDY_22171_sm_SuppFig1.tif11569KSupp. Fig. S1. CLMN is highly expressed in a small subset of large cells that line the periaqueductal gray in the adult rat brain. A–C: Three successively higher magnifications of this region are shown. Aqueduct (aq).
DVDY_22171_sm_SuppFig2.tif604KSupp. Fig. S2. Immunoblot analysis of CLMN protein in adult rat brain regions. A: The 160 kDa CLMN protein was detected using the CLMN antibody in rat brain regions and in the N2A cell control sample overexpressing CLMN3xFl (see arrowhead). Immunoblotting with nonimmune serum is shown as a control. The β-actin was also analyzed in (A) by immunoblotting as a control for protein loading. B: Densitometric analysis of the immunoblot shown in (A). CLMN levels were normalized to β-actin, with the highest CLMN expression found in the cortex. The next highest amounts were observed in the olfactory bulb, cerebellum, and hippocampus followed by the striatum. Brain regions were microdissected in homogenization buffer under a Nikon SMZ-U dissecting microscope and homogenized and processed as described in the Experimental Procedures section. Protein (20 μg) was resolved on a 7.5%/15% sodium dodecyl sulfate-polyacrylamide gel and probed with the CLMN antibody or nonimmune serum as described in the Experimental Procedures section. Membranes were blocked and incubated in 5% nonfat dry milk powder in phosphate buffered saline with 0.1% Tween-20 for the anti–β-actin antibody (Clone AC-15, Sigma, 1:5000). Protein levels were quantitated by densitometric analysis using the Personal Densitometer SI and ImageQuant 5.0 software (Molecular Dynamics, Sunnyvale, CA).
DVDY_22171_sm_SuppTable.doc31KSupplemental Table 1: HPLC-F9 reporter gene analysis of atRA levels in E10.5 rat embryos.

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