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: Electrophoretic mobility shift assay (EMSA) was performed using the LightShift Chemiluminescent EMSA Kit (WLA079a, Wanleibio, People's Republic of China), according to the manufacturer's protocol.
Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Isolation, Expressing, Plasmid Preparation, Transfection, Luciferase, Chromatin Immunoprecipitation
