ing4 polyclonal antibody  (Elabscience Biotechnology)

Journal: Turkish Journal of Pharmaceutical Sciences

Article Title: Evaluation of a Synthetic Polyethyleneimine Based Polymeric Vector for ING4 Gene Delivery to MCF-7 Breast Cancer Cells

doi: 10.4274/tjps.galenos.2023.72430

Figure Lengend Snippet: Protein expression of ING4 with western blot analysis

Article Snippet: Primary antibody incubation was performed using an ING4 polyclonal antibody from Elabscience (E-AB-33309), followed by Horseradish peroxidase (HRP)-conjugated secondary antibody incubation, both conducted in TBS-T containing 0.5% dry milk either at room temperature for 1 hour or at 4 ° C overnight.

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 interacted with nucleolar proteins. ( A ) The affinity assay was performed with recombinant GST-ING4 and the HeLa S3 cell lysate as described in the Methods. Candidate proteins from the silver-stained SDS-PAGE were identified by nanoLC/MS/MS system. Identified nucleolar proteins are listed on the table. ( B ) In vivo interaction between ING4 and NCL. The pull-down analysis with control IgG or anti-GFP antibody was conducted with the lysate of HAP1 cells stably expressing either GFP or GFP-ING4. Aliquots of lysates from cells that stably expressed GFP or GFP-ING4 were used as inputs. Bead-associated proteins were analyzed by the western blot with anti-nucleolin antibody. ( C ) In vivo interaction between ING4 and GNL3. Similar to ( B ), the lysate from HAPI cells was incubated with nanobody beads against GFP. The subsequent western blot was analyzed with anti-GNL3 antibody. ( D ) In vivo interaction between ING4 and NOLC1. Here, the lysate from HEK293T cells that temporarily expressed Flag or Flag-ING4 was incubated with anti-Flag antibody and immobilized with protein G agarose. Bead-associated proteins were analyzed by the western blot with anti-NOLC1 antibody ( E , F ) In vitro interaction between either GST-GNL3 ( E ) or GST-NOLC1 ( F ) and His-ING4. GST or GST-GNL3 or GST-NOLC1 beads were incubated with His-ING4. Elution was conducted with 10 mM reduced glutathione solution. Eluate (upper lane) and flow-through (lower lane) were evaluated using western blot with anti-His antibody for His-ING4.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Recombinant, Staining, SDS Page, Tandem Mass Spectroscopy, In Vivo, Stable Transfection, Expressing, Western Blot, Incubation, In Vitro

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: PHD domain was essential and sufficient for ING4 nucleolar localization. ( A ) Colocalization between GFP-ING4 and NCL in U-2 OS cells was evaluated by immunofluorescence study. Photos a, b, c, and d show GFP-ING4 in green, NCL in red, DAPI in blue and their merge, respectively. The data shown are the representative of three independent experiments with similar results. Scale bars in the figures indicate 10 µm. ( B ) GFP-fused ING4 domains that were used in the experiments were schematically shown. U-2 OS cells expressing various GFP-ING4 domains indicated in the photos were evaluated for GFP (green), NCL (red) and DAPI (blue). The data shown are the representative of three independent experiments with similar results. Scale bars in the figures indicate 10 µm.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Immunofluorescence, Expressing

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 positively regulated cell proliferation of HAP1 cells. ( A ) The expression of ING4 in the various types of HAP1 cell lysates was evaluated by the western blot analysis with anti-ING4 and anti-β-actin antibodies. Lane 1: wild type HAP1 cells, Lane 2: ING4-KO HAP1 cells, and Lane 3: ING4-KO HAP1 cells that were rescued by expression of GFP-ING4. ( B ) Three types of HAP1 cells described in ( A ) were seeded in 12-well plates at a density of 4 × 10 3 cells/well, then harvested on day 2, 4, and 8. The cell numbers were counted and displayed in the logarithm to the base 10. The data shown are means ±SD in three independent experiments. ** P < 0.01, *** P < 0.001. ( C ) HAP1 WT, KO and GFP-ING4-rescued KO cells used in this study were analyzed by the flow cytometry. Cell proliferation was analyzed by labelling with anti-BrdU antibody and DNA dye Propidium Iodide (PI). The flow cytometry analysis was based on 20,000 single-cell events. B: G 0 /G 1 population, C: G 2 /M population.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Expressing, Western Blot, Flow Cytometry

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 promoted the rRNA synthesis. ( A ) Equal amounts of total RNA extracted from WT, ING4-KO and GFP-ING4 rescued HAP1 cells were converted into cDNA, followed by RT-qPCR in triplicate. The relative pre-rRNA amounts were calculated by using the 2 −ΔΔCt method with β-actin normalization. The results shown are presented means ±SE in three independent experiments. ( B ) Similar experiments were performed with siRNA-mediated knockdown of ING4 in HAP1 cells. The upper insets display western blot results for ING4 or β-actin proteins. siCtrl: cells were transfected with negative control siRNA (Lane 1). siING4: cells were transfected with siRNA targeting ING4 at sequence #1 (Lane 2) or #2 (Lane 3). siRNA sequences were shown in Supplementary Table . The results shown are presented means ±SE in three independent experiments. * P < 0.05. ( C ) ING4 depletion reduced newly synthesized rRNA in HAP1 cells. HAP1 cells with/without 1-hour actinomycin D pre-treatment were incubated in a bundle of ribonucleotides including 5-BrUTP for 20 min before fixing for subsequent immunofluorescence. 5-BrUTP that incorporated into nascent rRNA was probed by Alexa Fluor 488-conjugated antibody and detected by confocal microscope. The full images were shown in Supplementary Information. ( D , E ) The specific consequence of ING4 depletion. ING4 expression levels had no effect on transcription ( D ) or protein ( E ) levels of several representative genes or proteins. Relative mRNA amounts were evaluated by RT-qPCR in normalization with β-actin. Protein levels were examined by western blot with specific antibodies as indicated in the figure.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Quantitative RT-PCR, Western Blot, Transfection, Negative Control, Sequencing, Synthesized, Incubation, Immunofluorescence, Microscopy, Expressing

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 enhanced H3K9 and histone H4 acetylation. ( A ) Levels of various modified histones were detected by western blot in three types of HAP1 cells, WT: wild type cells. KO: ING4-KO cells, KO + ING4: the GFP-ING4-rescued ING4-KO cells. Total histone H3 was used as the loading control. ( B – D ) The relative levels of H3K9ac ( B ), H3K4me3 ( C ) and H4ac ( D ) were evaluated by the densitometric analysis with the results of ( A ). The results shown are presented means ±SD in three independent experiments. * P < 0.05. ( E ) Levels of indicated proteins were detected by western blot in siRNA-treated HAP1 cells. siCtrl: cells were transfected with negative control siRNA. siING4: cells were transfected with siRNA targeting ING4 at sequence #1 or #2. Total histone H3 was used as the loading control. ( F , G ) The relative levels of H3K9ac ( F ) and H3K4me3 ( G ) in the western blot performed in ( E ) were quantified in the same way above. The results shown are presented means ±SD in three independent experiments. * P < 0.05.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Modification, Western Blot, Transfection, Negative Control, Sequencing

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 affected accumulation of histone modifications and UBF at the rDNA promoter. ( A ) The enrichment of H3K4me3, H3K9ac, H4ac and H4K16ac at the core promoter of rDNA was evaluated by ChIP/qPCR. qPCR was carried out in triplicate with chromatin DNA that was immunoprecipitated with the non-specific IgG or the specific antibodies. ( B ) The occupancy of UBF at enhancer (−1000 bp), core promoter (−50 bp) and transcript (+1000 bp) sites of rDNA was evaluated by ChIP/qPCR. It is noted that the transcription starting site is considered as +1. The primer sequences were provided in the Supplementary Table . The results shown are presented means ±SE in three independent experiments. ** P < 0.01 and *** P < 0.001.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Immunoprecipitation

Journal: Scientific Reports

Article Title: Inhibitor of Growth 4 (ING4) is a positive regulator of rRNA synthesis

doi: 10.1038/s41598-019-53767-1

Figure Lengend Snippet: ING4 levels regulated nucleolar morphology. ( A ) The localization of NCL in wild type (WT), ING4-KO (KO) and the GFP-ING4-rescued ING4-KO HAP1 (KO + ING4) cells were evaluated by the immunofluorescence study (left photos, in red). Nuclear staining (right photos, in blue) with Hoechst was also performed. The data shown are the representative of three independent experiments with similar results. Scale bars indicate 10 µm. ( B ) The proportion of cells with at least one ring-shaped nucleolus were calculated as described in the Methods. The data shown are means ±SD in 3 independent experiments. *** P < 0.001. ( C ) ING4-knockdown U-2 OS cells and the control U-2 OS cells were generated by transfecting with ING4-specific siRNA #1 or #2, and the negative control siRNA (siCtrl). Nuclei stained with Hoechst (left photos; in blue) and NCL (middle photos; in red) are shown. The photos on the right exhibited high magnification images of inset squares from the corresponding photos in the middle, (c) was from (b), (f) was from (e), and (i) was from (h). The data shown are the representatives of three independent experiments with similar results. Scale bars indicate 10 µm. ( D ) Numbers of NCL foci per cell were counted for at least 200 cells. The data shown are presented as means ±SE in 3 independent experiments. * P < 0.05.

Article Snippet: Anti-ING4 (C-term) polyclonal antibody was purchased from Abgent.

Techniques: Immunofluorescence, Staining, Generated, Negative Control

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