Journal: Nature Communications

Article Title: The NUCKS1-SKP2-p21/p27 axis controls S phase entry

doi: 10.1038/s41467-021-27124-8

Figure Lengend Snippet: a Schematic showing sequence positions of the EMSA probe in relation to SKP2 ’s transcription start site (TSS) and the region giving peak binding in our ChIP-qPCR assays. b Coomassie gel demonstrating NUCKS1 purification. Treatment with lambda phosphatase removes NUCKS1 phosphorylation and reduces its molecular weight. c Titration of phosphorylated or dephosphorylated NUCKS1 (10.24, 25.6, 64, 160, 400, 1000, 2500 nM) with the SKP2 promoter probe. d Quantification of c . e Titration of phosphorylated or dephosphorylated NUCKS1 with the SKP2 promoter probe. In lanes 6/12 and 7/13, respectively, 100 X molar quantity of unlabelled WT or mutant SKP2 probe were added as competition in binding reactions. f Quantification of e . g Titration of WT or NUCKS1 -KO U2OS nuclear extract with the SKP2 promoter probe. In lanes 6 and 11, 100 X molar quantity of unlabelled WT probe was added as competition in binding reactions. h Quantification of g . In b , c , e , and g , data are representative of 2 ( b ), 4 ( c ), or 3 ( e , g ) independent experiments. In d , f , and h , data are presented as mean ± SEM from 4 ( d ) or 3 ( f , h ) independent experiments. MW: molecular weight, kDa: kilodaltons. Source data are provided as a source data file.

Article Snippet: For NUCKS1 EMSAs, recombinant NUCKS1 was dephosphorylated using lambda phosphatase (100 units/1 μg of recombinant NUCKS1), in the presence of Protein MetalloPhosphatases buffer and MnCl 2 (1 mM), for 90 min at 30 °C, followed by addition of phosphatase inhibitor cocktail (50x, Merck Millipore).

Techniques: Sequencing, Binding Assay, ChIP-qPCR, Purification, Phospho-proteomics, Molecular Weight, Titration, Mutagenesis