Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Clinical and pathological data of patients of esophageal squamous cell carcinoma in this study

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: (a) BAP1 mutation in a patient with esophageal squamous cell carcinoma. In tumor tissue, TTT, encoding phenylalanine, is altered to ATT, encoding isoleucine (arrowhead). A faint peak observed in sequence data of this tumor likely represents contaminated residual non-cancerous DNA, as indicated by MLPA analysis (b). (b) MLPA data for all 17 exons of BAP1 gene on 3p21.1 are displayed on the x -axis. The table in the (b) also displays peaks of eight control probes, and RASSF1 MYND10 HEX1 ROBO1 FHIT MITF RBM5 and MLH1 in the BAP1 -neighboring region on chromosome 3p. Probe position and peak height in the (b) are described in SALSA MLPA probemix P417-B1 BAP1 (MRC Holland, Amsterdam, the Netherlands). Log 2 ratio of MLPA data for each probe is indicated on the y -axis. (c) Schematic of BAP1 with the F170I mutation. UCH, HCF1, ULD and NLS stand for ubiquitin C-terminal hydrolase domain, HCF1-binding domain, UCH37-like domain and nuclear localization signal, respectively. The F170I substitution is indicated by X. (d) Conservation of BAP1 in the region containing the F170I mutation. Alignment of amino acid sequences from codons 159–182 in human BAP1 and its counterparts in other species as identified by BLAST ( http://www.genome.jp/tools/blast/ ). Codon 170 is boxed.

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis, Sequencing, Control, Ubiquitin Proteomics, Binding Assay

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: (a) Deubiquitination of HCF1 by BAP1. Plasmids as described were transfected into HEK-293T cells. Cell extracts were immunoprecipitated with anti-HA antibody, followed by western blot analysis with either anti-Myc or with anti-HA antibody. The expression level of HCF1 or BAP1 (wild-type or F170I mutant) was confirmed by western blot analyses with anti-HA or anti-FLAG antibody. (b) Statistical analysis in triplicate, ubiquitination of HCF1. Statistical significance is indicated by the asterisk ( P < 0.05, Student's t -test). (c) Deubiquitination of BAP1. Plasmids were transfected into HEK-293T cells. Cell extracts were immunoprecipitated with anti-BAP1 antibody, followed by western blot analysis with either anti-Myc or anti-BAP1. (d) Statistical analysis in triplicate, ubiquitination of BAP1. Statistical significance is indicated by the double asterisk (P < 0.01, Student's t -test).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Ubiquitin Proteomics

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Subcellular localization of wild-type BAP1 or F170I mutant. Exogenous BAP1, wild-type (Wt) or F170I mutant (F170I), was visualized by anti-FLAG antibody treatment, followed by incubation with Alexa Fluor 488-conjugated antibody. A representative result is shown in (a). Exogenous BAP1 was more highly expressed in the nucleus (N > C), expressed equally between the nucleus and the cytoplasm (C = N), or expressed more highly in the cytoplasm (C > N). In each sample at least 200 cells were counted (b).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Mutagenesis, Incubation

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Heat map analyses of gene expression profiles. MT and WT stand for F170I mutant and wild-type BAP1, respectively. Expression profiles were examined in two independent transfections, 1 or 2 (a). A total of 5840 genes, selected by principal component analysis from the values of the first component of the vector, excluding genes without genome annotation, were used for heat map analysis by adjusting the average expression level of each gene to 0 (b).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Gene Expression, Mutagenesis, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Immunohistochemical staining of BAP1 in surgically resected esophageal cancer tissues. Representative features are shown, under low magnification (upper; bar 50 μm) and high magnification (lower; bar 20 μm). We classified (a) as BAP1 negative, (b) as BAP1-positive. In eight nuclear BAP1-negative cases, BAP1 was strongly expressed within the cytoplasm (c).

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Immunohistochemical staining, Staining

Journal: Cancer Science

Article Title: Somatic alteration and depleted nuclear expression of BAP1 in human esophageal squamous cell carcinoma

doi: 10.1111/cas.12722

Figure Lengend Snippet: Result of quantitative RT-PCR for TCEAL7 expression in wild-type BAP1 or F170I mutant BAP1-transfected HEK-293T cells. RT-PCR was repeated in triplicate for duplicated transfections. Each asterisk indicates statistical significance ( P < 0.001). TF1, TF2 and NS stand for transfection 1, transfection 2 and no statistical significance ( P = 1.0 between TF1 and TF2 by F170I; P = 0.43 between TF1 and TF2 by Wt), respectively.

Article Snippet: Subsequently, the slides were incubated at 41°C overnight with anti-BAP1 antibody (sc-28383, Santa Cruz, diluted 1/100).

Techniques: Quantitative RT-PCR, Expressing, Mutagenesis, Transfection, Reverse Transcription Polymerase Chain Reaction