rabbit anti ing4  (Cell Signaling Technology Inc)

Journal: Oncology Letters

Article Title: Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma

doi: 10.3892/ol.2017.6450

Figure Lengend Snippet: ING4 mRNA is significantly decreased in CCRC tissues when compared with normal renal tissues. (A) A total of 40 paired tumor and adjacent normal renal tissues were analyzed for ING4 mRNA level by reverse transcription-quantitative polymerase chain reaction. β-actin was used as an internal control. (B) Analysis of relative mRNA level (compared with normal renal tissue) according to Fuhrman nuclear grade (24) (grade I, n=7; grade II, n=16; grade 3, n=13; grade 4, n=4), lymphatic metastasis (n0, n=29; n1, n=7; n2, n=4), tumor clinical stage (stage I, n=9; stage II, n=12; stage III, n=14; stage IV, n=5). ***P<0.001 by t-test. CCRC, clear cell renal carcinoma; ING4, inhibitor of growth family member 4.

Article Snippet: Briefly, 4-µm-thick sections were blocked with 5% bovine serum albumin in PBS prior to incubation overnight at 4°C with rabbit anti-human ING4 polyclonal antibody (1:100; cat no. 40-7700; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma

doi: 10.3892/ol.2017.6450

Figure Lengend Snippet: ING4 expression is significantly decreased in CCRC tissues when compared with normal renal tissues. (A) A total of 40 paired tumor and adjacent normal renal tissues were analyzed for ING4 expression using western blotting. Decreased ING4 expression was observed in CCRC tissues compared with adjacent normal renal tissues. β-actin was used as internal control. (B) Average ING4 expression in 40 paired tumor and normal renal tissues samples. (C) Analysis of the expression level of ING4 protein (compared with normal renal tissue) according to tumor nuclear grade (grade I, n=7; grade II, n=16; grade III, n=13; grade IV, n=4), lymphatic metastasis (n0, n=29; n1, n=7; n2, n=4), tumor clinical stage (stage I, n=9; stage II, n=12; stage III, n=14; stage IV n=5). ***P<0.001 by t-test. CCRC, clear cell renal carcinoma; ING4, inhibitor of growth family member 4; NT, normal tissue.

Article Snippet: Briefly, 4-µm-thick sections were blocked with 5% bovine serum albumin in PBS prior to incubation overnight at 4°C with rabbit anti-human ING4 polyclonal antibody (1:100; cat no. 40-7700; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing, Western Blot

Journal: Oncology Letters

Article Title: Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma

doi: 10.3892/ol.2017.6450

Figure Lengend Snippet: ING4 protein expression detected using the EnVision system in (A) normal renal tubular epithelial cell nuclei, and in renal cell carcinoma; (B) nuclear grade I, cellular expression; (C) nuclear grade II, cell membrane and cytoplasmic expression; (D) nuclear grade III, cytoplasmic expression; (E) nuclear grade IV, cytoplasmic expression; (F) positive expression of cytoplasmic granules. Magnification of all images, ×400. ING4, inhibitor of growth family member 4.

Article Snippet: Briefly, 4-µm-thick sections were blocked with 5% bovine serum albumin in PBS prior to incubation overnight at 4°C with rabbit anti-human ING4 polyclonal antibody (1:100; cat no. 40-7700; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma

doi: 10.3892/ol.2017.6450

Figure Lengend Snippet: ING4 protein in different nuclear grade renal cell carcinoma tissues.

Article Snippet: Briefly, 4-µm-thick sections were blocked with 5% bovine serum albumin in PBS prior to incubation overnight at 4°C with rabbit anti-human ING4 polyclonal antibody (1:100; cat no. 40-7700; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques:

Journal: Oncology Letters

Article Title: Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma

doi: 10.3892/ol.2017.6450

Figure Lengend Snippet: ING4 protein expression in patients with clear cell renal carcinoma of the association between the clinical and pathological features.

Article Snippet: Briefly, 4-µm-thick sections were blocked with 5% bovine serum albumin in PBS prior to incubation overnight at 4°C with rabbit anti-human ING4 polyclonal antibody (1:100; cat no. 40-7700; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Expressing

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: ( A ) Determination of ING4 protein levels in 10 cancer tissues and paired non-cancerous normal colon tissues by western blot. ( B ) Real time PCR showed the differences of the raw delta Ct values of ING4 amplification after normalization by GAPDH in cancers and paired normal tissues. ( C ) Representative images of ING4 immunohistochemical staining in TMAs were shown. Note: Top panel: original magnification, × 50; bottom panel: original magnification, × 200. ( D ) The distribution of the difference in staining intensities of ING4 in CRC tissues compared with that in paired normal tissues. C, CRC tissues; N, paired non-cancerous colon tissues. ( E ) Kaplan–Meier curves showed overall survival of CRC according to expression levels of ING4.

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Amplification, Immunohistochemical staining, Staining, Expressing

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: Relationship between the expression level of ING4 and clinicopathological features of CRC patients

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Expressing

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: Univariate Cox regression analysis of ING4 expression and clinicopathological variables predicting the survival of CRC patients

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Expressing

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: Multivariate Cox regression analysis models assessing the effects of covariates on OS in CRC patients

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Expressing

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: ( A – B ) Western blot was used to test the ING4 expression in the p53 +/+ HCT116 and HCT15 cells with ING4 overexpression (HA-ING4), ING4 knockdown (mi-ING4) and respective vector control (HA-Vec and mi-Vec). ( C ) ING4 in CRC cells negatively regulated tube formation. ( D – E ) Numbers of complete tubular structures formed by HUVECs were counted for ING4 overexpressed, knocked down and control groups ( n = 3/group) in CRC cells. ( F ) Photographs of matrigel plugs with ING4 overexpressed or control p53 +/+ HCT116 and HCT15 cells excised from mice after 10 days of growth in vivo . Data are presented as means ± standard deviations. * P < 0.05, ** P < 0.001 (Student's t -test).

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Western Blot, Expressing, Over Expression, Plasmid Preparation, In Vivo

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: ( A ) Western blot confirmed that ING4 inhibited the expression of nuclear Sp1. ( B ) ING4 altered the DNA affinity of Sp1. EMSA was performed using nuclear protein extracts from p53 +/+ HCT116 transfected with ING4 overexpression (HA-ING4), knockdown (mi-ING4) and respective controls plasmids (HA-Vec and mi-Vec). Lane 1 contains no nuclear extracts. All other lanes contain 1μg nuclear extracts. Lane 6 represents competition analysis using 100-fold unlabeled Sp1 probes. The super shift band was observed when the Sp1 antibody was added (lane 7) and IgG was used as negative control for super shift (lane 8). ( C – D ) Real time PCR and western blot were used to explore the expressions of Sp1 downstream pro-angiogenic genes MMP-2 and COX-2 in ING4 over-expressed, knocked down and control p53 +/+ HCT116 cells. ( E ) The increased expressions of MMP-2 and COX-2 by ING4 knockdown were abolished by Sp1 siRNA (si-Sp1). ( F ) Conditioned medium was collected and applied in tube formation. ( G ) Numbers of complete tubular structures formed by HUVECs were counted for ING4 knockdown, Sp1 knockdown or co-knockdown and control groups in p53 +/+ HCT116 ( n = 3/group). ( H ) Photographs of matrigel plugs with ING4 knockdown, Sp1 knockdown or co-knockdown and control p53 +/+ HCT116 cells excised from mice after 10 days of growth in vivo . ( I ) The expressions of ING4, Sp1, MMP-2 and COX-2 were examined by western blot in matrigel plugs containing ING4 knockdown, Sp1 knockdown or co-knockdown and control p53 +/+ HCT116 cells. Data are presented as means ± standard deviations. * P < 0.05, ** P < 0.001 (Student's t -test).

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Western Blot, Expressing, Transfection, Over Expression, Negative Control, Real-time Polymerase Chain Reaction, In Vivo

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: ( A – B ) ING4 overexpression (HA-ING4) significantly enhanced but ING4 knockdown (mi-ING4) decreased the degradation of Sp1. p53 +/+ HCT116 cells were transfected with ING4 overexpression, knockdown and respective controls plasmids 48 hours, respectively, followed by exposure to cycloheximide (CHX; 50 mg/ml) for 0, 4, 8 or 12 hour, and the whole-cell lysates were detected by western blot. ( C – D ) The intensity of the Sp1 protein bands was analyzed by densitometry, after normalization to the corresponding beta-actin level. ( E ) Ubiquitination of Sp1 was induced by ING4. The p53 +/+ HCT116 cells were transfected with GFP-ub together with HA-Vec or HA-ING4 plasmids for 48 hour and then pretreated with MG132 (10 μM) for 6 hour, and these whole-cell lysates were immunoprecipitated using anti-Sp1 antibody, and ubiquitination was detected with ubiquitin antibody. Endogenous Sp1 were examined by western blot. ( G ) The intensity of the Sp1 ubiquitin bands was analyzed by densitometry. The data are means ± standard deviations from three independent experiments. * P < 0.05, ** P < 0.001 (Student's t -test).

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Over Expression, Transfection, Western Blot, Immunoprecipitation

Journal: Oncotarget

Article Title: ING4 suppresses tumor angiogenesis and functions as a prognostic marker in human colorectal cancer

doi: 10.18632/oncotarget.12984

Figure Lengend Snippet: ( A ) ING4 increased p21 expression. The expressions of cell-cycle associated proteins, such as cyclin A, cyclin E, CDK2, p21 and p27 in the p53 +/+ HCT116 cells with ING4 overexpression (HA-ING4) or knockdown (mi-ING4) and the respective controls (HA-Vec and mi-Vec). ( B ) ING4 regulated expressions of p21 and Sp1 regardless of p53 status. The expressions of p53, p21, Sp1 and histone H1, a sensitive protein for cyclin/CDK2 complexes phosphorylation activity, in the p53 +/+ HCT116 and p53 −/− HCT116 cells with ING4 overexpression or knockdown and the respective controls. ( C ) ING4 regulated p21 mRNA expression. The mRNA expressions of p21 in the p53 +/+ HCT116 and p53 −/− HCT116 cells with ING4 overexpression or knockdown and the respective controls was determined by real time PCR. ( D ) ING4 regulated p21 promoter activity. The reporter gene assay was used to test the p21 promoter activity in the p53 +/+ HCT116 and p53 −/− HCT116 cells with ING4 overexpression or knockdown and the respective controls. ( E ) ING4 regulated p21 to mediate Sp1 expression. The expressions of p21 and Sp1 were tested in the p53 +/+ HCT116 cells transfected with p21 overexpression (GFP-p21) together with ING4 knockdown and the control plasmids. ( F ) The model of ING4 suppressing tumor angiogenesis through p21/cyclin/CDK2 complexes/Sp1/MMP-2 and COX-2 signaling pathway. Data are presented as means ± standard deviations. * P < 0.05, ** P < 0.001 (Student's t -test).

Article Snippet: The polyclonal rabbit anti-ING4 antibody (1:2000 dilution, Sigma, USA) was used for primary antibody incubation at 4°C overnight.

Techniques: Expressing, Over Expression, Activity Assay, Real-time Polymerase Chain Reaction, Reporter Gene Assay, Transfection

Journal: PLoS ONE

Article Title: MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

doi: 10.1371/journal.pone.0072615

Figure Lengend Snippet: ( A ) Western blot analysis of ING4 protein expression in docetaxel-resistant and parental LAD cells. ( B ) Sequence of miR-650 binding site in the ING4 3’ UTR predicted with TargetScan, miRBase and PicTarget and the 3’-UTR region of ING4 mRNA is partially complementary to miR-650. ( C ) SPC-A1/DTX or H1299/DTX cells were co-transfected with miR-650 mimics or inhibitor and pLUC vector with ING4 3’-UTR-wt or mut. After 24 hours, the luciferase activity was measured. Values are presented as relative luciferase activity after normalization to Renilla luciferase activity. ( D ) Western Blot analysis of ING4 protein expression in SPC-A1/DTX or H1299/DTX cells transfected with miR-650 mimics (or miR-NC mimics) or anti-miR-650 (or anti-miR-NC). The data are expressed as the mean value ± SEM of the results obtained from three independent experiments. * P <0.05 or ** P <0.01, in comparison with anti-miR-NC or miR-NC mimics-transfected cells.

Article Snippet: Paraffin-embedded, formalin-fixed tissues were immunostained for ING4 using a rabbit anti-ING4 polyclonal antibody (Santa Cruz) at 1:500 dilution.

Techniques: Western Blot, Expressing, Sequencing, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Comparison

Journal: PLoS ONE

Article Title: MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

doi: 10.1371/journal.pone.0072615

Figure Lengend Snippet: ( A ) Western blot analysis of ING4 protein expression in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. ( B ) MTT analysis of growth in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. * P <0.05, in comparison with anti-miR-650-transfected cells or cells co-transfected with anti-miR650 and siRNA/NC. ( C ) Analysis of the IC 50 values of docetaxel in anti-miR-NC or anti-miR650-transfected SPC-A1/DTX cells or SPC-A1/DTX cells co-transfected with anti-miR650 and siRNA/NC or siRNA/ING4. ( D ) Western Blot analysis of ING4 protein expression in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4. ( E ) MTT analysis of growth in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4. * P <0.05, in comparison with miR-650 mimics-transfected cells or cells co-transfected with miR-650 mimics and pcDNA/control. ( F ) Analysis of the IC 50 values of docetaxel in miR-NC or miR-650 mimics-transfected SPC-A1 cells or SPC-A1 cells co-transfected with miR-650 mimics and pcDNA/control or pcDNA/ING4 using MTT assay. The data are expressed as the mean value ± SEM of the results obtained from three independent experiments.

Article Snippet: Paraffin-embedded, formalin-fixed tissues were immunostained for ING4 using a rabbit anti-ING4 polyclonal antibody (Santa Cruz) at 1:500 dilution.

Techniques: Western Blot, Expressing, Transfection, Comparison, Control, MTT Assay

Journal: PLoS ONE

Article Title: MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

doi: 10.1371/journal.pone.0072615

Figure Lengend Snippet: ( A ) Flow cytometric analysis of apoptosis in anti-miR-NC or anti-miR-650-transfected SPC-A1/DTX cells. ( B ) Hoechst staining analysis of apoptosis in anti-miR-NC or anti-miR-650-transfected SPC-A1/DTX cells. ( C ) Western blot detection of the expression of pro-caspase-3 and cleaved caspase-3 proteins in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). ( D ) Detection of caspase-3 activity in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). ( E ) Western blot analysis of the expression of Bcl-2 and Bax proteins in anti-miR-650 or pcDNA/ING4-transfected SPC-A1/DTX cells (anti-miR-NC or pcDNA/control was used as control). Equal loading was confirmed by showing equal GAPDH levels. Results represent the average of three independent experiments (mean±SD). * P <0.05 or ** P <0.01, in comparison with anti-miR-NC or pcDNA/control-transfected cells.

Article Snippet: Paraffin-embedded, formalin-fixed tissues were immunostained for ING4 using a rabbit anti-ING4 polyclonal antibody (Santa Cruz) at 1:500 dilution.

Techniques: Transfection, Staining, Western Blot, Expressing, Control, Activity Assay, Comparison

Journal: PLoS ONE

Article Title: MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

doi: 10.1371/journal.pone.0072615

Figure Lengend Snippet: ( A ) qRT-PCR analysis of miR-650 expression in SPC-A1/DTX cells stably transfected with pLMP-miR-NC or pLMP-miR-650. ( B ) Analysis of the docetaxel IC 50 values in stably transfected SPC-A1/DTX cells. ( C ) Western blot analysis of ING4 protein expression in stably transfected SPC-A1/DTX cells. ( D ) SPC-A1/DTX cells stably expressing miR-650 were injected into mice subcutaneously. After the tumor was established (tumor size=50 mm 3 ), mice were i.p. injected with a concentration of 1.0 mg/kg (one dose every other day with 3 doses totally) followed by monitoring of tumor size for 5 weeks (mean±SEM, n=8). * P < 0.05.

Article Snippet: Paraffin-embedded, formalin-fixed tissues were immunostained for ING4 using a rabbit anti-ING4 polyclonal antibody (Santa Cruz) at 1:500 dilution.

Techniques: Quantitative RT-PCR, Expressing, Stable Transfection, Transfection, Western Blot, Injection, Concentration Assay

Journal: PLoS ONE

Article Title: MicroRNA-650 Was a Prognostic Factor in Human Lung Adenocarcinoma and Confers the Docetaxel Chemoresistance of Lung Adenocarcinoma Cells via Regulating Bcl-2/Bax Expression

doi: 10.1371/journal.pone.0072615

Figure Lengend Snippet: ( A ) Immunohistochemical staining for ING4 protein in non-responding or responding LAD tissues. Isotype IgG at the same concentration was used as a negative control for immunohistochemical staining. Positive ING4 protein staining was mainly located in the nucleus of tumor cells. IN4 expression was higher in responding tumors than in nonresponding tumors. Bars : 100µm. ( B ) Relative expression levels of ING4 protein was detected in docetaxel-responding (n=25) and non-responding (n=19) LAD tissues via Western blot assay. GAPDH was used as an internal control. ( C ) A statistically significant inverse correlation between miR-650 and ING4 protein levels in 44 cases of LAD tissues (Spearman’s correlation analysis, r = -0.0645; P =0.018). Results represent the average of three independent experiments (mean±SD). Corresponding P values analyzed by Spearman correlation test are indicated.

Article Snippet: Paraffin-embedded, formalin-fixed tissues were immunostained for ING4 using a rabbit anti-ING4 polyclonal antibody (Santa Cruz) at 1:500 dilution.

Techniques: Immunohistochemical staining, Staining, Concentration Assay, Negative Control, Expressing, Western Blot, Control