non-responsive reporter pg15-luc  (Addgene inc)

Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Interaction of p53 and WT1 proteins. Detection of WT1-p53 protein interactions by immunoprecipitation and western blot analysis. HeLa cells were transfected with expression plasmids encoding the WT1-GFP-fusion proteins, wild-type WT1, mutant WT1 Wilms3 and mutant WT1 Wilms2 . As control, cells were transfected with a GFP-expression plasmid alone. Lysates from transfected cells were incubated with control antibodies (lanes, αCtrl) or p53-specific antibodies (lanes, αP53). Immunoprecipitates were resolved by SDS–PAGE and immunoblotted using antibodies specific for the N-terminus of the WT1 protein (αF6WT1). As input controls, 5% of the respective lysates from transfected HeLa cells were loaded on the gel (lanes, In). Asterisk indicates a non-specific band. Wild-type and mutant WT1-GFP-fusion proteins in the input and immunoprecipitated fractions are marked by black boxes.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Mutagenesis, Control, Plasmid Preparation, Incubation, SDS Page

Journal: Human Molecular Genetics

Article Title: Classification of a frameshift/extended and a stop mutation in WT1 as gain-of-function mutations that activate cell cycle genes and promote Wilms tumour cell proliferation

doi: 10.1093/hmg/ddu111

Figure Lengend Snippet: Identification of p53 networks in cohorts of WT1 Wilms3 target genes. The MetaCore program ‘What are the key transcription factors and target genes in my data’ was used to identify p53 networks in subsets of genes whose expressions are either down-regulated ( A ) or up-regulated ( B ) by sh WT1 knockdown in Wilms3 cells. Blue circles mark down-regulated genes ( A ), and red circles mark up-regulated genes ( B ). The properties of individual genes are indicated by coloured symbols. These symbols are explained in Supplementary Material, Table S15 . Green arrows point to genes that are activated by p53 and red arrows to genes that are repressed by p53.

Article Snippet: Endogenous p53 activity in Wilms3 cells was measured with the p53-responsive reporter pG13- Luc [13 p53 binding sites, Addgene, 16 442 ( )] and the non-responsive reporter pG15- Luc [15 mutant p53 binding sites, Addgene, 16 443 ( )].

Techniques: Knockdown