Fig 1: The effects of FATS-262D/N on p53 ubiquitination and activation. a Purified GST protein or GST-tagged FATS protein was incubated with purified E1 and ubiquitin protein in ubiquitination buffer at 30 C for 90 min. The polyubiquitination was examined by Western blot using an ubiquitin-specific antibody (n = 3). FATS-262 N exhibited stronger E3 activity than did FATS-262D in assembling ubiquitin chains. b Purified GST protein or GST-tagged FATS protein was incubated with purified E1, ubiquitin and p53 protein in ubiquitination buffer at 30 °C for 90 min. The non-proteolytic polyubiquitination was examined by Western blot using a p53-specific antibody (n = 3). c MCF-7 cells were transfected with indicated vectors. Luciferase reporter assay was performed in triplicate in three independent experiments after transfection for 24 h. The pGL2-p21-luc vector contains a p21 promoter with p53-responsive elements. d MCF-7 cells were transfected with Flag-tagged FATS-262D or FATS-262 N or an empty vector, respectively. After 24 h, cells were treated with etoposide (25 µM) for the indicated time. Cell lysates were subjected to immunoblotting, and the results assessed quantitatively (n = 3). Ac, acetylation; Phos, phosphorylation
Fig 2: Distribution of major SNP sites in FATS locus and validation of rs11245007. a The illustration of genomic organization of human FATS locus. b The distribution of SNP sites in FATS exons. MAF: minor allele frequency. c Validation of rs11245007 genotypes in Chinese population. d Sequence alignment of amino acid residues within RING/HECT hybrid domain between human FATS and mouse FATS. The conserved catalytic Cys residue of FATS as a unique E2-independent E3 is highlighted
Fig 3: Clinical relevance of FATS expression in breast cancer. a Quantitative analysis of FATS mRNA (GenBank accession number: NM_001004298) in breast cancer. Tumor samples and paired (>5 cm away) normal breast tissue samples (n = 38) from the Tianjin cohort were subjected to RNA extraction and subsequent RT-PCR analysis. The average CT value for FATS gene in each sample was obtained from three independent experiments, and normalized by that of GAPDH gene to obtain ?CT. The quantity of FATS mRNA in each sample was calculated as 2-?CT. b Immunohistochemistry of FATS in paired human breast tumor samples and normal breast tissue samples (n = 30). A representative picture is shown with the same magnification (40X). N, normal; T, Tumor. c The average level of FATS mRNA from three independent experiments in breast tumor samples (n = 156) from the Tissue Bank Facility of TMUCIH was profiled according to pathological stages, in comparison with that in normal breast tissues. N: normal (n = 38); T-I: TNM stage I (n = 40); T-II: TNM stage II (n = 97); T-III: TNM stage III (n = 19). *, P < 0.05; **, P < 0.01
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