In vitro Protein Binding Assays

Proteins were produced using TNT™ Quick Coupled /Translation System (Promega) according to the manufacturer's instructions. Probe annealing and experiments were carried out as described before (41 (link)) for mouse DMRT1 and with some modifications for GATA4 and SOX9. For SOX9 binding assay, the non-specific competitor dI-dC was replaced by poly(dG-dC) (GE Healthcare). Mouse GATA4 was in vitro translated using as template the pCS2⁺mGATA4 plasmid, a generous gift from Dr Sergei Tevosian.
For Figure 4, the top strand probes were:
Lats2: 5΄-ggCCGAGCGGGACATTCGCTACATTGTTGGCATTCCACGGGCG-3΄
Lmo4: 5΄-ggCATCTCCCATTATTGTTCCAAATCTCATTTCATTTTGAA-3΄
Mrpl45: 5΄-ggGTTTTTTTCACCGATTGTAAATAAGGTGTAACAATGTGTTAAGGAACCAGGA-3΄
DMRT1 BS (12 (link)): 5΄-ggGGGAGATTTGATACATTGTTACTTTATGG-3΄
GATA4 BS (42 (link)): 5΄-ggGGGGCTTTGGTCTCAGCTTATCAAACTGCCCTG-3΄
SOX9 BS (43 (link)): 5΄-ggGTTGACAGAACAATGGCTGCTAGA-3΄
The ‘g’ nucleotides at C-terminus were added for probe labelling with Klenow DNA polymerase and α [³²P]-dCTP.

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Rahmoun M., Lavery R., Laurent-Chaballier S., Bellora N., Philip G.K., Rossitto M., Symon A., Pailhoux E., Cammas F., Chung J., Bagheri-Fam S., Murphy M., Bardwell V., Zarkower D., Boizet-Bonhoure B., Clair P., Harley V.R, & Poulat F. (2017). In mammalian foetal testes, SOX9 regulates expression of its target genes by binding to genomic regions with conserved signatures. Nucleic Acids Research, 45(12), 7191-7211.

Publication 2017

TNT™ Quick Coupled /Translation System

Assay Dctp Dmrt1 Dna polymerase Lats2 Mouse Nucleotides Plasmid Poly Promega Proteins Sox9

Corresponding Organization :

Other organizations : Institut de Génétique Humaine, Hôpital Arnaud de Villeneuve, Hudson Institute of Medical Research, Inserm, Institut de Recherche en Cancérologie de Montpellier, Centro Científico Tecnológico - Patagonia Norte, Melbourne Bioinformatics, Laboratoire de Biologie du Développement, Twin Cities Orthopedics, University of Minnesota, Laboratory of Pathogens and Host Immunity, Institut de Génomique Fonctionnelle, Montpellier GenomiX

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Variable analysis

independent variables

Type of probe used for binding assay (e.g., Lats2, Lmo4, Mrpl45, DMRT1 BS, GATA4 BS, SOX9 BS)

dependent variables

Protein binding to the various probe sequences

control variables

Experimental protocol for probe annealing and binding assays, as described in the provided references
Use of the non-specific competitor poly(dG-dC) for the SOX9 binding assay, instead of the commonly used dI-dC

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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