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

Journal: Frontiers in Fungal Biology

Article Title: Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance

doi: 10.3389/ffunb.2021.689900

Figure Lengend Snippet: A. fumigatus ZnfA:3xHA is not dephosphorylated upon calcium stress. ZnfA:3xHA strain was grown for 24 h at 37°C and treated or not with CaCl 2 10 mM for 10 min. Protein extracts were subjected to immunoprecipitation and left untreated or treated with lambda phosphatase (λ-PP). Bidimensional gel electrophoresis coupled with western blotting was then carried out by using anti-HA antibody against the HA epitope to detect ZnfA:3xHA. pI, isoelectric point.

Article Snippet: For the lambda protein phosphatase treatment, the ZnfA:3xHA immunoprecipitates bound to magnetic beads were incubated with PMP buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, and 1 mM MnCl 2 ) and 400 units of lambda protein phosphatase (New England BioLabs) (without phosphatase inhibitor cocktail) for 1 h at 30°C.

Techniques: Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot

Journal: Frontiers in Fungal Biology

Article Title: Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance

doi: 10.3389/ffunb.2021.689900

Figure Lengend Snippet: ZnfA binds to the promoter regions of specific genes encoding for siderophore biosynthesis and transport and cell wall remodeling after caspofungin exposure. Chromatin immunoprecipitation coupled to DNA sequencing (ChIP-seq) of the ZnfA:3xHA strain when grown for 16 h at 37°C and treated or not with 2 μg/ml of CSP for 1 h was assessed. (A) Heat map depicting ChIP-seq signal for genes involved in siderophore biosynthesis and transport and cell wall organization. (B) Selected examples of peaks identified in the promoter region of genes belonging to the siderophore biosynthetic cluster, zfpA and znfA genes in control and CSP (2cas) conditions. (C) DNA sequence and E-value of the putative ZnfA binding motif identified by MEME-ChIP analysis, that was enriched in the presence of CSP. (D) Heatmap showing normalized expression ratios of selected genes whose promoter regions were bound by ZnfA in the presence of CSP (2 μg/ml) according to the ChIP-seq analysis. The heatmap graph was created by using GraphPad Prism 8. Statistical analysis was performed using one-tailed, paired t -tests for comparisons between the control and caspofungin condition for the wild-type strain (Wild-type vs. Wild-type+CSP; black asterisks) and both strains in the presence of the drug (Wild-type+CSP vs. Δ znfA +CSP; white asterisks) (E) Radial growth phenotypes were determined for the wild-type, Δ znfA and Δ znfA::znfA + strains after 5 days of growth at 37°C in MM, MM-Fe, and MM-Fe supplemented with FeSO 4 200 μM with and without CSP. Standard deviations represent averages of results from three independent biological repetitions. Statistical analysis was performed using one-tailed, paired t -tests for comparisons to the control condition (MM plus CSP) ( * P < 0.05; ** P < 0.01).

Article Snippet: For the lambda protein phosphatase treatment, the ZnfA:3xHA immunoprecipitates bound to magnetic beads were incubated with PMP buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, and 1 mM MnCl 2 ) and 400 units of lambda protein phosphatase (New England BioLabs) (without phosphatase inhibitor cocktail) for 1 h at 30°C.

Techniques: Chromatin Immunoprecipitation, DNA Sequencing, ChIP-sequencing, Sequencing, Binding Assay, Expressing, One-tailed Test

Journal: Frontiers in Fungal Biology

Article Title: Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance

doi: 10.3389/ffunb.2021.689900

Figure Lengend Snippet: List of the main ZnfA:3xHA target genes identified in the ChIP-seq analysis enriched in the caspofungin exclusive condition.

Article Snippet: For the lambda protein phosphatase treatment, the ZnfA:3xHA immunoprecipitates bound to magnetic beads were incubated with PMP buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM DTT, 0.01% Brij 35, and 1 mM MnCl 2 ) and 400 units of lambda protein phosphatase (New England BioLabs) (without phosphatase inhibitor cocktail) for 1 h at 30°C.

Techniques: Expressing, Activity Assay, Mutagenesis, Sequencing