Fig. 1: Temporal and spatial expression pattern of RNF166 mRNA and protein in various tissues.
a In situ hybridization assay was performed with [35S]-labeled RNA probes encoding antisense sequences of mouse RNF166 encompassing 1~500 bp. Various planes of section were examined to better visualize the expression pattern of RNF166 mRNA at the indicated stages of development: sagittal sections of the whole body at E16 and E18; horizontal sections of the mouse brain at postnatal day 7 (P7), P14, and P21; horizontal (H), coronal (C), and sagittal (S) sections of 2-month-old adult brain. For negative controls, [35S]-labeled RNA probes encoding sense sequences of RNF166 were hybridized with midsagittal sections of E18 embryos and adult mouse brain. The following abbreviations are used from left to right of the antisense panel: NC neopallial cortex, VZ ventricular zone, RM roof of midbrain, SC spinal cord, Cb cerebellum, Cx cerebral cortex, OB olfactory bulb, Hc hippocampus, STR striatum. Photomicrographs represent data acquired from RNA probe #1. Virtually identical patterns were obtained using RNA probes #2 and #3. b Total RNA prepared from various tissues of adult mice were subjected to RT-PCR. As an internal control, universal 18S primers were included in the same reaction mixtures. c Tissue lysates were prepared from the various body parts of adult mice and subjected to immunoblotting using anti-RNF166 antibody. Anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. d Tissue lysates obtained from various brain parts of adult rats were subjected to immunoblotting using anti-RNF166 antibody. Densitometric values of RNF166 normalized to GAPDH are expressed as a percentage of that in the cerebellum (value = 100%). Bars represent the mean + SEM of three independent experiments. ****p < 0.0001. e Temporal expression levels of RNF166 determined using tissue lysates obtained from rat cerebral cortex at E16, P7, postnatal week 3 (W3), and W8. Densitometric values of RNF166 signals were normalized against actin and are expressed as percentage relative to that in E16 cortices (value = 100%). Bars represent the mean + SEM from three independent experiments. ****p < 0.0001.
