biotin labeled probe  (General Biosystems Inc)

Journal: Horticulture Research

Article Title: PtrbZIP12 improves drought resistance in Populus trichocarpa by directly targeting PtrDHN and PtrPOD

doi: 10.1093/hr/uhag034

Figure Lengend Snippet: PtrbZIP12 directly activates the promoters of PtrDHN and PtrPOD . (a) Relative expression levels of PtrDHN and PtrPOD in PtrbZIP12 -OE versus WT plants. Data are presented as means ± SD from three biological replicates. (b) Verification of the direct regulatory interaction between PtrbZIP12 and the PtrDHN promoter using ChIP-PCR, in which the promoter region (−800 to −1 bp) was divided into four fragments (P1–P4). ‘Input’ represents chromatin before immunoprecipitation, ‘ChIP-’ indicates immunoprecipitation without antibody, and ‘ChIP+’ denotes immunoprecipitation with anti-GFP antibody. (c) Y1H assay demonstrating PtrbZIP12 binding to ABRE motifs; controls included p53-HIS2/pGADT7-Rec2-p53 (positive) and pGADT7-Rec2- PtrbZIP12 /p53-HIS2 (negative). (d and e) Y1H assays showing PtrbZIP12 binding to the promoters of PtrDHN and PtrPOD , with P53-promoter-AUR1-C and AD-Rec-P53 as positive controls, and AD-empty prey vector with AUR1-C driven by the target gene promoter as negative controls. (f) EMSA confirming PtrbZIP12 –ABRE binding; lanes: 1, biotin-labeled probe; 2, labeled probe + PtrbZIP12 protein; 3–5, competition with 10-, 50-, and 100-fold molar excess of unlabeled probe. (g and h) Schematic representation of effector and reporter constructs utilized in the dual-LUC assay, with transient LUC/renillase (REN) coactivation experiments in N. benthamiana leaves demonstrating PtrbZIP12 -mediated activation of PtrDHN (g) and PtrPOD (h).

Article Snippet: Specific biotin-labeled DNA probes targeting the promoter region of the gene were synthesized by Sangon Biotech Co.

Techniques: Expressing, Immunoprecipitation, Y1H Assay, Binding Assay, Plasmid Preparation, Labeling, Construct, Activation Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Genetic Regulation of Wnt/PCP Components Through Fgf10/Fgfr2/Sox9 Module in Tear Duct Development

doi: 10.1167/iovs.67.3.30

Figure Lengend Snippet: Sox9 binds and activates Prickle1 promoter. ( A, B ) Verified Sox9 binding consensus sequence ( S0 , above ) and a 4 kb-upstream regulatory region from the Prickle1 transcription starting site (TTS) with four predicted sequences of Sox9 binding variants ( S1-4 , under ). “ cs ” stands for complementary strand. ( B ) Oligonucleotides of wild type ( S1-4 ) and mutants ( mt-S1-4 ). ( C ) Electrophoretic mobility shift assay (EMSA) to test Sox9 binging oligos that were illustrated in ( B ). “ bio-oligos ”, biotin-labeled wild type oligonucleotides ( S0-S4 ); “ cold-oligos ”, unlabeled wild type oligonucleotides; “ mt-oligos ”, unlabeled mutant oligonucleotides; “Sox9-extr.”, nuclear extracts isolated from Sox9-expressing vector-transfected 661W cells; “Ctrl-extr.”, nuclear extracts from empty vector transfected 661W cells; “+”, with; “ – ”, without. Shifted bands are enclosed within the rectangular box with asterisks indicating gels. The arrow points to the free probes. Only S3 gel showed an expected shift that can be competed away with wild type but not mutant unlabeled probes. Experimental details refer to “Materials and Methods” section. ( D ) Luciferase assays testing basal-level transcription activities of Prickle1 promoters containing neither TATA box nor predicted Sox9 binding sites (P0), TATA box alone (P1), and TATA box and all predicted binding sites (P2). ( E ) In the presence of Sox9, promoter P2 showed significantly elevated transcription activities, which was markedly reduced when S3 was mutated ( mt-S3 ). ( F ) Chromatin immunoprecipitation assay (ChIP) enriched S3 -containing PCR products ( left ), and Gapdh promoter by RNA pol II ( right ).

Article Snippet: Double-stranded biotin-labeled DNA probes containing the putative binding motif were synthesized by Sangon Biotech (People's Republic of China).

Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Isolation, Expressing, Plasmid Preparation, Transfection, Luciferase, Chromatin Immunoprecipitation