cdna for ncor1  (Transomic Technologies Inc)

Journal: Nature neuroscience

Article Title: NCOR1/2 loss of function impairs memory through a novel GABAergic hypothalamus–CA3 projection

doi: 10.1038/s41593-018-0311-1

Figure Lengend Snippet: . (a) Patients carrying copy number variants (CNV) or single nucleotide variants (SNV) in NCOR1 , NCOR2 , or HDAC3 . Genomic coordinates are shown in hg19. DDD_SNV, single nucleotide variants retrieved from Deciphering Developmental Disorders (DDD) website (United Kingdom); kb, kilobase. (b-d) Schematic representations for the deletions and point mutations affecting NCOR1 , NCOR2 , or HDAC3 , respectively, observed in patients with neurodevelopmental disorders. The locations of deletions are depicted in red, and the point mutations in pink. (e) Western blot of HEK-293 cells transfected with plasmids expressing wild-type (WT) HDAC3 with or without mutant L266S. The experiment was repeated independently once with similar results. The blot images have been cropped. (f) Fluorescence-based HDAC enzyme assay after anti-HDAC3 immunoprecipitates from cell lysates overexpressing the indicated HDAC3 proteins. Box plots center line, median; box limits, upper and lower quartiles; whiskers, minimal and maximum values. Data were analyzed by two-tailed unpaired t test. n=4 biological independent samples for each group. (g) Western blot of HEK-293 cells transfected with plasmids expressing WT HDAC3, WT NCOR1, with or without the NCOR1 deletion mutant (Del). The experiment was repeated independently once with similar results. Data were analyzed by two-tailed unpaired t test. n=3 biological independent samples for each group. The blot images have been cropped. (h) Chromatin immunoprecipitation (ChIP) with anti-HDAC3 antibodies followed by qPCR using primers targeting promoters of the indicated genes ARNTL and CDKN1A . RPLP0 serves as a negative control. Data is expressed as mean ± S.E.M. For detailed statistics results, see . * P ≤ 0.05 is set as significance.

Article Snippet: Human full-length cDNA for NCOR1 was obtained from transOMIC and was sub-cloned into the pcDNA3.1-Zeo+ plasmid with C-terminal Flag-tag using standard PCR method for both WT and the 1-666 truncation. pcDNA3-based Flag-tagged HDAC3 cDNA construct was described before and the L266S point mutation was introduced using standard PCR method.

Techniques: Western Blot, Transfection, Expressing, Mutagenesis, Fluorescence, Enzymatic Assay, Two Tailed Test, Chromatin Immunoprecipitation, Negative Control