Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 1: Comparison of the expression levels of BAP1, OGT and YY1 genes in the tumor group and the control group.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Comparison, Expressing, Control

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 2: ROC curves of the levels of BAP1, OGT and YY1 in patients with eyelid tumors.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques:

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 3: BAP1(A–B), OGT (D–E), and YY1 (G–H) immunolocalization in BCC. Negative control (C–F–I). White arrow: positive cells in tumor area and black arrow: positive cells in stromal cells. Chromogen; AEC, contrast dye; Hematoxylin. The intensity of the proteins was determined at ×10, ×20 and ×40 magnification with semi-quantitative scoring.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Negative Control

Journal: Turkish Journal of Biochemistry

Article Title: Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors

doi: 10.1515/tjb-2021-0160

Figure Lengend Snippet: Figure 4: BAP1, OGT and YY1 staining scores in BCC tumor and control areas.

Article Snippet: To prevent non-specific binding, sections were incubated for 10 min in Ultra V Block (Thermo Scientific, USA) at RT, and then treated with anti-BAP1 antibody (rabbit polyclonal IgG, EPP10790, Elabscience, USA) (1:100 dilution), anti-OGT antibody (rabbit polyclonal IgG, EAP0752, Elabscience, USA) (1:100 dilution), and anti-YY1 antibody (rabbit polyclonal IgG, EPM12542, Elabscience, USA) (1:100 dilution) overnight at +4 °C.

Techniques: Staining, Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 6. Western blotting analysis of tight junction protein expression in the lungs of fetal rats. (A, B) Representative immunoblotting and densitometric analysis of tight junction protein expression in the fetal lung. Results were normalized relative to the expres- sion of β-actin (n = 6 per group, *P < 0.05, vs. con- trol). Western blot analysis showed increased levels of Cldn3 and decreased levels of Magi1 and Myh9 in the CDH fetal lungs.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Western Blot, Expressing

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 7. IHC analysis of tight-junction proteins in the lungs of fetal rats. (A) Representative photomicrographs of IHC-staining for Cldn3, Magi1, and Myh9 in the lung sections from CDH (Left panel) and Control (right panel) fetal rats. (Original magnification ×400, scale bar = 100 μm). Cldn3 and Magi1 were mainly expressed in the epithelial cells, and some weak staining was also found in the mesenchymal cells. Myh9 protein localized to both epithelial and mesenchymal cells. (B) Semi‑quantitative analysis of IHC staining (mean density). n = 3 per group, *P < 0.05, vs. control.

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Immunohistochemistry, Control, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

doi: 10.1016/j.biopha.2019.109621

Figure Lengend Snippet: Fig. 8. Temporal expression of tight junction mRNA in CDH lungs. (A–C) Cldn3, Magi1, and Myh9 mRNA levels in CDH lungs were determined by quantitative RT- PCR at different time points. β-actin was used as a housekeeping gene. The results were normalized relative to the same-aged control group (n = 8 per group, *P < 0.05, vs. control at the same time point).

Article Snippet: The membranes were blocked in 5% nonfat dry milk for 60min and then incubated with rabbit polyclonal anti-CLDN3 (Proteintech, Chicago, USA), rabbit polyclonal anti-MAGI1 antibody (Absin, Shanghai, China), rabbit polyclonal anti-MYH9 (Proteintech Group, Chicago, USA), and mouse monoclonal β-actin (Proteintech Group, Chicago, USA) overnight at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: Identification of HCF-1 as a Bap1 interacting protein. (A) Schematic comparing structures of human UCH family members: Bap1 (RefSeq accession no. NP_004647), UCH37 (NP_057068), UCHL1 (NP_004172), and UCHL3 (CAG33136). The Bap1 UCH domain is most similar to that of UCH37 (42.5% sequence identity) and includes the critical cysteine, histidine, and aspartate residues of the active site. Bap1 and UCH37 share an additional region of homology at the C terminus (ULD, green box). In UCH37, this region folds as a coiled coil that interacts with the Rpn13 subunit of the proteasome. (B) Sequences of the C-terminal homology region (ULD) from human and mouse Bap1 (accession numbers NP_004647 and NP_081364) and UCH37 (accession numbers NP_057068 and AAD31534). Residues in UCH37 that are identical (filled) or similar (shaded) to Bap1 are indicated. (C) Purification of Flag-tagged Bap1 (Bap1-Flag). Silver-stained SDS-PAGE gel (4 to 12% gradient) showing eluted proteins after anti-Flag coimmunoprecipitation from a 1% digitonin lysate of Bap1-Flag-expressing 293 cells. Bands from a Bap1-Flag sample and corresponding regions of the control sample were excised, digested with trypsin, and subjected to peptide analysis by MS/MS. In three independent experiments, multiple bands in the 100-kDa or larger size range were identified as HCF-1 from the Bap-1 elution but not the control.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Sequencing, Purification, Staining, SDS Page, Expressing, Tandem Mass Spectroscopy

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: HCF-1 associates with Bap1 in transfected cells. (A) Sequences of 27 HCF-1-derived peptides identified by MS/MS from proteins coimmunoprecipitated with Bap1-Flag. The amino acid positions in the prototype human HCF-1 protein sequence (RefSeq accession no. NP_005325) are indicated. (B) Coassociation of Bap1-Flag and HCF-1-V5 in transfected cells. Bap1-Flag and HCF-1-V5 were expressed individually or together in human 293 cells, and their expression was confirmed in immunoblotting of the 1% digitonin lysates (left panels). Bap1-Flag was immunoprecipitated using Flag antibody and precipitated proteins detected by immunoblotting with V5 or Flag (right panels). (C) Bap1 and HCF-1 colocalize within the cell. HeLa cells were transiently transfected with a Bap1-HA expression plasmid, fixed and subjected to indirect immunofluorescence using an α-HA monoclonal antibody to detect Bap1 and anti-HCF-1 (N18) polyclonal antibody to detect endogenous HCF-1. Scale bar, 5 μm. (D) Bap1-Flag associates with endogenous HCF-1 both in cytoplasm and nucleus. Whole-cell lysates (T) or cytoplasmic (C) and nuclear (N) fractions were prepared from control 293 cells or cells stably expressing low levels of Bap1-Flag and probed by immunoblotting either directly or after immunoprecipitation with anti-Flag beads. Sp1 serves as a nuclear protein control.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Transfection, Derivative Assay, Tandem Mass Spectroscopy, Sequencing, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation, Immunofluorescence, Stable Transfection

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: The β-propeller domain of HCF-1 is sufficient for interaction with Bap1. (A) Schematic representation of HCF-1 to indicate the positions of the β-propeller, basic region, HCFPRO repeats (arrowheads), transactivation domain (AD), and tandem Fn3 repeats. Four HCF-1 truncations used in panel B have been described previously (43) and correspond to HCF-1Δrep (NC), HCF-1N1011 (N), HCF-1N380 (Nβ), and HCF-1N450-1011 (Nbasic). An HA-epitope tag is included at the N terminus of each HCF-1 polypeptide. (B) The HCF-1 β-propeller domain is sufficient for interaction with Bap1. Plasmids encoding Bap1-Flag and selected HA-tagged HCF-1 truncations were transiently transfected into 293 cells, and their association was examined by coimmunoprecipitation. Cells were lysed in 1% digitonin and Bap1-Flag immunoprecipitated from the clarified lysates using anti-Flag beads. Recovered proteins were detected by immunoblotting anti-HA and anti-Flag antibodies. (C) Catalytic activity of Bap1 is not required for association with endogenous HCF-1. Control 293 cells (mock) or cells stably expressing low levels of wild-type (Wt) or active site mutant (C91A) Bap1-Flag were lysed in 1% digitonin and immunoprecipitated using anti-Flag beads. Endogenous HCF-1 was detected by immunoblotting with anti-HCF-1 antibody.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Transfection, Immunoprecipitation, Western Blot, Activity Assay, Stable Transfection, Expressing, Mutagenesis

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: Association with HCF-1 requires a conserved tetrapeptide HBM in the central region of Bap1. (A) An HBM-like sequence is located near the center of Bap1, within a region that is highly conserved in vertebrate Bap1 sequences. Accession numbers are as follows: human (Homo sapiens), NP_004647; mouse (Mus musculus), NP_081364; chicken (Gallus gallus), NP_001025761; zebrafish (Danio rerio), XP_687254; and African clawed toad (Xenopus laevis), NP_001089388. (B) Mutation of the NHNY sequence abrogates the interaction with HCF-1. Empty Flag vector (lanes 1 and 5) or vectors encoding wild-type Bap1-Flag (lanes 2 and 6), C91A Bap1-Flag (lanes 2 and 6), or NHNY/AAAA Bap1-Flag (lanes 4 and 8) were transiently cotransfected into 293 cells, together with either empty Myc tag vector (lanes 1 to 4) or HCF1-Myc vector (lanes 5 to 8). Cells were lysed in buffer containing 1% digitonin, immunoprecipitated with anti-Flag antibody beads, and coprecipitated HCF-1-Myc detected by immunoblotting with anti-Myc antibody. (C) The NHNY/AAAA Bap1 mutant retains enzymatic activity. Immunopurified Flag-tagged proteins were washed extensively, eluted from the beads with free three-Flag peptide, and assayed for the ability to cleave a ubiquitin-AMC fluorogenic substrate. Purified UCH-L3 and cells transfected with empty vector served as positive and negative controls. Values correspond to the average of three independent assays, and the standard deviations are shown.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Sequencing, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Activity Assay, Purification, Transfection

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: Catalytically inactive Bap1 promotes the selective accumulation of K48 ubiquitinated HCF-1. (A) HCF-1 is ubiquitinated through both lysine-48 (K48) and lysine-63 (K63) linkages. 293 cells were transiently transfected with plasmid encoding HCF-1-V5 and HA-tagged K48 or K63-ubiquitin. Cells were treated for 2 h with 20 μM MG132 prior to lysis in NP-40-containing buffer supplemented with 1% SDS. After removal of the insoluble material, the lysates were diluted in 10 volumes of buffer lacking SDS, and V5-tagged HCF-1 was recovered by anti-V5 immunoprecipitation. Linkage of ubiquitin moieties was detected by immunoblotting with anti-HA antibody. (B) Ectopic expression of Bap1 alters the level of HCF-1 K48 ubiquitination. Plasmids encoding HCF-1-V5, K48 HA-Ub, and wild-type or C91A mutant Bap1-Flag were introduced into 293 cells and processed as described for panel A. (C) Bap1 expression only affects levels of K48 ubiquitinated HCF-1. HCF-1-V5, C91A Bap1-Flag, K48 HA-Ub, or K63 HA-Ub were expressed as indicated in 293 cells. Prior to lysis, cells were treated for 2 h with 20 μM MG132. HCF-1-V5 was immunoprecipitated (NP-40 + 1% SDS lysis, followed by 10-fold dilution in buffer without SDS) with anti-V5 antibody, and equal amounts of HCF-1-V5 were loaded on gels, followed by Western blotting with anti-HA antibody. The results show that levels of K48 ubiquitinated HCF-1 are considerably increased when C91A Bap1 is expressed; however, no noticeable effects on the levels of K63 ubiquitinated HCF-1 were observed.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Transfection, Plasmid Preparation, Lysis, Immunoprecipitation, Western Blot, Expressing, Mutagenesis

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: Endogenous HCF-1 is ubiquitinated, and the major site of HCF-1 ubiquitination is mapped to K1807 or K1808 residues. (A) Scheme for partial purification of ubiquitinated HCF-1 through association with Bap1-Flag. (B) Samples from Bap1-Flag-expressing or control 293 cells were subjected to sequential anti-Flag and anti-HA immunoprecipitation as summarized for panel A and analyzed by MS/MS. Peptide masses and fragment ions characteristic of two tryptic peptides derived from HCF-1C were monitored throughout an LC-MS/MS experiment. Three fragment ions for each peptide were detected in the samples from Bap1-Flag-expressing cells but not the controls. (C) Purification of HCF-1-V5 for ubiquitination site mapping. HA-Ub was expressed in 293 cells with or without HCF-1-V5. HCF-1-V5 polypeptides were recovered from denatured lysates by immunoprecipitation with anti-V5 beads. After extensive washing, bound proteins were eluted and detected by immunoblotting with anti-V5 and anti-HA antibodies (left panel) or directly visualized by silver staining (right panel). (D) Identification of lysine-1807 and/or lysine-1808 of HCF-1C as major sites of ubiquitination. Eluted HCF-1-V5 and control samples (from C) were analyzed by MS/MS, and a single HCF-1 peptide was identified to be ubiquitinated. Highlighted fragment ions are derived from HCF-1C peptide 1804-APMKKENQWFDVGVIK-1809 and display a mass shift indicative of a ubiquitin linkage at lysine-1807 and/or lysine-1808.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Purification, Expressing, Immunoprecipitation, Tandem Mass Spectroscopy, Derivative Assay, Liquid Chromatography with Mass Spectroscopy, Western Blot, Silver Staining

Journal:

Article Title: Association of C-Terminal Ubiquitin Hydrolase BRCA1-Associated Protein 1 with Cell Cycle Regulator Host Cell Factor 1 ▿

doi: 10.1128/MCB.01517-08

Figure Lengend Snippet: Bap1 contributes to HCF-1 stability. (A) Depletion of Bap1 using siRNA. Control 293 cells or cells stably expressing Bap1-Flag were treated with control or Bap1 siRNA for 3 days. Endogenous and ectopic Bap1 were immunoprecipitated and blotted with a rabbit polyclonal antibody 419 against Bap1 (α-Bap1). (B) Depletion of Bap1 leads to a modest stabilization of HCF-1. 293 cells were treated with control siRNA or Bap1 siRNA for 2 days and then transfected with HCF-1-V5. After 48 h, cells were lysed, and the clarified lysates were probed by immunoblotting with anti-V5 antibody. Blotting for tubulin served as a loading control. (C) Depletion of Bap1 alters cell cycle progression. Subconfluent HeLa cells were cotransfected with a GFP expression plasmid and either a Bap1 siRNA or a control siRNA. After 48 h, cells were fixed and stained with propidium iodide, and GFP-positive cells were analyzed for DNA content by flow cytometry. For easier comparison of the two FACScalibur profiles, a dotted line marks the 4N peak in the mock siRNA sample. (D) Schematic showing the proposed association of Bap1 with the two subunits of HCF-1. Bap1 contains a nonconsensus HBM that is recognized by the β-propeller domain of the HCF-1N subunit and required for association of Bap1 with HCF-1. By analogy to UCH37, the C-terminal coiled-coiled (C-C) region of Bap1 may facilitate recruitment to the proteasome, whereas the UCH domain catalyzes the removal of K48-linked ubiquitin moieties. A candidate substrate might be the HCF-1C subunit, which is shown to be ubiquitinated at conserved lysine-1808 and/or lysine-1809 in the first Fn3 repeat.

Article Snippet: Rabbit polyclonal anti-Bap1 antibody was raised against a synthetic polypeptide corresponding to Bap1 residues 322 to 436 (Yenzym Antibodies LLC).

Techniques: Stable Transfection, Expressing, Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation, Staining, Flow Cytometry