Journal: Development (Cambridge, England)

Article Title: miR-126-5p promotes retinal endothelial cell survival through SetD5 regulation in neurons.

doi: 10.1242/dev.156232

Figure Lengend Snippet: Figure 8: SetD5 requires BRD2 to control Sema3A expression

Article Snippet: Complexes were immunoprecipated in the same conditions than for in vitro experiments using 1μg of SetD5 antibody or IgG control and BRD2 immunoblotting analysis were performed with Novus rabbit anti-BRD2 antibody (NBP1-30475).

Techniques: Control, Expressing

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: The target antigen of the XC246 autoantibody was identified as BRD2. (A) Preparative 10% SDS-PAGE was performed to isolate the XC246 antigen, and in-gel digestion was carried out for mass spectrometric-based protein identification. A preparative SDS-PAGE gel for western blotting was divided into two sections and blotted separately. The western blotting result is a combined image of two blots, with a dotted line representing the edges of two images. The protein band containing the XC246 antigen confirmed by western blotting was excised (indicated by the red arrow) and in-gel digested with trypsin. The proteins identified by mass spectrometric analysis are listed in . (B) Validation of the XC246 antigen as BRD2 by an RNA interference assay. HepG2 cells were transfected with siRNAs for candidate genes (EEF2, MYO1C and BRD2), and their cell lysates were examined by western blotting with the XC246 antibody. The knockdown of target genes was confirmed using reverse transcription polymerase chain reaction or western blotting. GAPDH was used as an internal control. (C) Immunoprecipitation analysis for the verification of the XC246 antigen as BRD2. The HepG2 cell lysate was immunoprecipitated with XC246 antibody-conjugated agarose beads and analyzed by western blotting with an anti-BRD2 or the XC246 antibody. Immunoprecipitates obtained using agarose beads without antibody conjugation were used as the control. Red arrows indicate the XC246 antigen or BRD2. (D) Immunofluorescence staining of the XC246 antigen in HepG2 cells. Fixed and permeabilized cells were treated with purified XC246 antibody or an anti-BRD2 antibody, followed by staining with FITC- or RDM-labeled anti-mouse IgG. To visualize the nuclei, cells were stained with DAPI. To verify the nuclear permeability of stained cells, an IgM-type mouse antibody (FBXO2 antibody) was also employed. (E) Western blot analysis of the intracellular distribution of the XC246 antigen or BRD2. Total cell lysates, subcellular fractions (cytosolic or nuclear fractions), and exosome lysates were prepared as described in the 'Materials and methods' and analyzed using western blotting. The blots were probed with the XC246 autoantibody, anti-BRD2 antibody, or anti-ATIC antibody. Each target antigen is indicated by colored arrows (red: XC246 and exosome XC246 antigen; blue: BRD2; green: ATIC). BRD2, bromodomain-containing protein 2; RDM, rhodamine; ATIC, AICAR transformylase/inosine monophosphate cyclohydrolase.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: SDS Page, Western Blot, Biomarker Discovery, Transfection, Knockdown, Reverse Transcription, Polymerase Chain Reaction, Control, Immunoprecipitation, Conjugation Assay, Immunofluorescence, Staining, Purification, Labeling, Permeability

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Mass spectrometric analysis of XC246 antigen.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques:

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: BRD2 autoantibody ELISA was developed using XC246p9 epitope-conjugated BSA for the detection of autoantibodies in human sera. (A) Biopanning of a phage-display random cyclic heptapeptide library for the isolation of epitope mimicries of the XC246 antigen. (B) Phage ELISA to confirm the binding specificity of the selected epitope mimicry phages to the XC246 autoantibody. M13 phages were coated with 10 11 pfu phages per well. Primary antibodies were used at the concentration of 0.1 µ g/well. Unrelated autoantibodies [K94 and XC90 ] were used as non-related controls. (C) Competitive FACS analysis of XC246 autoantibody binding to XC246 phages or HepG2 cells. Fixed and permeabilized cells (1×10 5 cells/reaction) were treated with the XC246 autoantibody (0.5 µ g). For antibody binding competition with the selected phages, cells were treated with the XC246 antibody pre-incubated with each phage (10 11 or 10 12 pfu/reaction), as indicated. (D) Preparation of the BSA-miniPEG2-XC246p9 antigen. A cyclic peptide with two miniPEG spacers, miniPEG2-XC246p9, was chemically synthetized and conjugated to bovine serum albumin (BSA) via amine-carboxyl acid coupling using the EDC reagent. The peptide-BSA conjugates (5 µ g/lane) were analyzed by SDS-PAGE and Coomassie blue staining. BSA-miniPEG2 without the epitope peptide was prepared as a control antigen. The synthetic peptide-conjugated to BSA was observed as a high-molecular-weight protein band. (E) ELISA with the BSA-miniPEG2-XC246p9 antigen. The antigen was coated at the indicated amount and detected with a gradually diluted XC246 autoantibody. (F) Competitive western blot analysis of XC246 autoantibody binding to the BSA-miniPEG2-XC246p9 antigen or tumor cell lysates. The cell lysates [HepG2 (H) or SNU638(S)] were loaded at a quantity of 15 µ g per lane, and BRD2 was detected with the XC246 autoantibody (1 µ g/10 ml). For the competitive inhibition of antibody binding to cell lysates, the XC246 antibody was pre-incubated with the BSA-miniPEG2-XC246p9 antigen (0.6 µ g/ml). BSA-miniPEG2-XC90p2 or BSA was used as the control competitor. The XC90p2 sequence is as follows: CPVRSGFPC. GAPDH was used as a loading control. BRD2, bromodomain-containing protein 2; BSA, bovine serum albumin.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Binding Assay, Concentration Assay, Incubation, SDS Page, Staining, Control, High Molecular Weight, Western Blot, Inhibition, Sequencing

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Human serum BRD2 autoantibody ELISA using BSA-miniPEG2-XC246p9 differentiated patients with HCC from non-HCC subjects. (A) AFP test and BRD2 autoantibody ELISA using BSA-miniPEG2-XC246p9 in sera of patients with HCC, as well as non-tumor subjects. The sample distribution was as follows: Control (n=91), HCC (n=118), cirrhosis (n=32) and benign liver cancer (n=3). Serum AFP levels were measured using a commercial quantification kit. The CV of AFP was 20 ng/ml, and the proportion of AFP-positive or -negative HCC (APHC or ANHC) is indicated within the box. The specific binding of the serum autoantibody to the XC246p9 epitope (anti-BRD2 response) was described as the difference in OD between the ELISA with BSA-miniPEG2-XC246p9 and that with BSA-miniPEG2. (B) The ROC curve analysis revealed the diagnostic sensitivity and specificity of each biomarker. All experiments were performed in duplicate and repeated at least three times. (C) Serum AFP and BRD2 autoantibody response related to tumor stage. The non-HCC group included control, cirrhosis, and benign liver cancer samples. (D) Serum AFP and BRD2 autoantibody response related to viral infection. The clinicopathological features of the participants are described in detail in . ns, not significant (P>0.05); BRD2, bromodomain-containing protein 2; BSA, bovine serum albumin; HCC, hepatocellular cancer; CV, cut-off value; APHC, AFP-positive HCC; ANHC, AFP-negative HCC; AFP, serum alpha-fetoprotein.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Binding Assay, Diagnostic Assay, Biomarker Discovery, Infection

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: Combined analysis of serum autoantibody biomarkers with AFP enhanced the diagnostic accuracy of HCC. (A) Pearson's analysis of the correlations between the BRD2 autoantibody biomarker and the AFP or ATIC autoantibodies. The dotted lines represent the cutoff value of each biomarker diagnosis. The results of Pearson's analysis in individual cohorts are depicted in Fig S10. (B) Combined analysis of HCC biomarkers, AFP, BRD2 autoantibody, or ATIC autoantibody. The diagnostic values of each biomarker shown in panel A (AFP, anti-BRD2, and anti-ATIC) were simplified as either S-1 or S-0 according to whether their detection values surpassed or fail the cut-off value. Subsequently, the diagnostic values of each biomarker or their combination were analyzed. For the combined analysis of these markers, the unified diagnostic indexes of a serum sample were simply added and designated triple-negative samples as S-0, single-positive samples as S-1, double-positive samples as S-2, and triple-positive samples as S-3. The numbers on the plots represent the percentage of corresponding subjects. (C) Scattered plot analysis of HCC biomarker responses depending on AFP and autoantibody biomarker. The numbers in each quadrant represent the percentage of each case among patients with HCC (n=118). The numbers in the parentheses are the proportion of autoantibody biomarker-positive or -negative samples among ANHC or APHC cases. HCC, hepatocellular cancer; BRD2, bromodomain-containing protein 2; AFP, serum alpha-fetoprotein; ATIC, AICAR transformylase/inosine monophosphate cyclohydrolase; S-1, responsive; S-0, non-responsive; S-2, double positive; S-3, triple positive; ANHC, AFP-negative HCC; APHC, AFP-positive HCC.

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Diagnostic Assay, Biomarker Discovery

Journal: International Journal of Oncology

Article Title: Serum BRD2 autoantibody in hepatocellular carcinoma and its detection using mimotope peptide-conjugated BSA

doi: 10.3892/ijo.2022.5448

Figure Lengend Snippet: The clinicopathological features of the validation cohort a .

Article Snippet: The primary antibodies used in this study were as follows: BRD2 (Novus Biologicals, cat. no. NBP1-84310, NBP1-30475; 1:1,000 dilution), AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC; Thermo Fisher Scientific, cat. no. MA1-086; 1:500 dilution), programmed cell death 6-interacting protein (ALIX; exosomal marker; Merck Millipore, cat. no. ABC1435; 1:500 dilution), calnexin (endoplasmic reticulum marker; Santa Cruz Biotechnology, cat. no. sc-46669; 1:1,000 dilution), GAPDH (Santa Cruz Biotechnology, cat. no. sc-47724; 1:5,000 dilution), and β-actin (Santa Cruz Biotechnology, cat. no. sc-8432; 1:5,000 dilution).

Techniques: Biomarker Discovery, Infection, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Pharmacologic Targeting of BET Proteins Attenuates Hyperuricemic Nephropathy in Rats

doi: 10.3389/fphar.2021.636154

Figure Lengend Snippet: I-BET151 reduces Brd4 levels in the kidney of HN rats. (A) A rat model of HN was established and treated with I-BET151 as indicated in “Material and Methods.” After 3 weeks, kidneys were taken for immunoblot analysis for Brd2, Brd3, Brd4 or GAPDH. Expression levels of Brd2 (B) , Brd3 (C) , Brd4 (D) were quantified by densitometry analysis and then normalized with GAPDH. (E) Photomicrographs (original magnification, ×400) illustrate immunohistochemical Brd4 staining of kidney tissues. (F) Brd4 staining graphic presentation of quantitative data. Data are represented as the mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: Brd2 antibody was purchased from Boster Biological Technology (Wuhan, China), OAT1 and OAT3 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), I-BET151 was purchased from Target Mol (MA, United States).

Techniques: Western Blot, Expressing, Immunohistochemical staining, Staining

Journal: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 induces degradation of BRD2, BRD3, and BRD4 in HCC cells. (A) HepG2 cell line was treated by BETd-260, HJB-97, or JQ1 as indicated for 24 h. The protein levels of BRD2, BRD3 and BRD4 were examined by western blot analysis. Actin was used as a loading control. (B) HepG2 cell line was treated by BETd-260 at 100 nmol/L for different times. The protein levels of BRD2, BRD3, and BRD4 were examined by western blot analysis. Actin was used as a loading control. (C) BEL-7402, SK-HEP-1, SMMC-7721, HuH-7, and MHCC97H cell lines were treated by BETd-260 at 100 nmol/L for 24 h. The protein levels of BRD2, BRD3 and BRD4 were examined by western blot analysis. Actin was used as a loading control. Data are representative of three independent experiments.

Article Snippet: The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Targeting BET Proteins With a PROTAC Molecule Elicits Potent Anticancer Activity in HCC Cells

doi: 10.3389/fonc.2019.01471

Figure Lengend Snippet: BETd-260 induces BET degradation, triggers apoptosis, and inhibits proliferation in HCC xenograft tissue in mice. BALB/c mice bearing HepG2 and BEL-7402 xenograft tumors were treated by a single intravenous dose of 5 mg/kg BETd-260 (BETd) for 24 h or vehicle (Veh). (A) Two to three mice were sacrificed and tumor tissue was harvested. The expression of BRD2, BRD3, and BRD4, Mcl-1, Bad, activated caspase-3, cleaved PARP, and Ki 67 was examined by immunohistochemistry staining. Representative photographs were presented. (B) The percentages of HCC tumor cells positively stained with BRD2, BRD3, and BRD4, Mcl-1, Bad, cleaved caspase-3 (c-Casp3), cleaved PARP (c-PARP), and Ki 67 were quantified under microscopy, and plotted. Data are representative of three independent experiments. ** p < 0.01.

Article Snippet: The following antibodies were used for IHC: BRD2 (IHC-00612), BRD4 (HC-00396), BAD (A302-384A) from Bethyl Laboratories (Shanghai, China); BRD3 (ab264420) from Abcam (Shanghai, China); cleaved PARP (Asp214) (#32563), activated caspase-3 (#9664), and Ki-67 (8D5) (9449) from Cell Signaling Technology (CST, Shanghai, China), Anti-Mcl-1 (MAB828) from R&D Systems (R&D Systems, Shanghai, China).

Techniques: Expressing, Immunohistochemistry, Staining, Microscopy