Knockdown of ERG was performed as previously described (Hogan et al., 2017 (link)) using 1 nM siRNA oligonucleotides in OptiMEM (Thermo Fisher Scientific, Cat# 11058021) with Lipofectamine 2000 (Thermo Fisher Scientific, Cat# 11668030). Transfections were performed in serum-free media for 4 hr, then cells were grown in full growth media for 48 hr. All siRNAs and qPCR primers used in this study are listed in Supplementary file 1r . Transfection efficiency for the siRNAs utilized in this study was verified using qPCR 7 d after transfection (Figure 3—figure supplement 3A ). Protein knockdown is shown 2 d after transfection using the same siRNAs from a representative experiment (Figure 3—figure supplement 3B ). Antibodies used included 1:1000 recombinant anti-ERG antibody (ab133264) and 1:5000 anti-histone H3 antibody (ab1791) (Abcam). Western blots were quantified using ImageJ (Schneider et al., 2012 (link)).
Ab133264
Manufactured by Abcam
Sourced in Denmark, Hungary, United States
Ab133264 is a primary antibody product manufactured by Abcam. It is designed for use in various laboratory techniques.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Ab1791, by Abcam (2 mentions)
Af3639, by R&D Systems (2 mentions)
Ab199428, by Abcam (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
A008229, by Agilent Technologies (1 mentions)
Anti β actin hrp, by Merck Group (1 mentions)
Ab4055, by Abcam (1 mentions)
Quantcenter image analysis software, by 3DHISTECH (1 mentions)
Pannoramic viewer, by 3DHISTECH (1 mentions)
Anti gapdh mab374, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Goat anti mouse igg dylight 800, by Thermo Fisher Scientific (1 mentions)
Anti akt 11e7, by Cell Signaling Technology (1 mentions)
Anti dll4, by R&D Systems (1 mentions)
Anti nicd cleaved notch1 val1744, by Cell Signaling Technology (1 mentions)
Ab23980, by Abcam (1 mentions)
Ab7029, by Abcam (1 mentions)
Ab32631, by Abcam (1 mentions)
10 protocols using ab133264
1
Knockdown of ERG Protein Using siRNA
2
Western Blot Analysis of Vascular Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as previously described [27 ]. Primary antibodies used were rabbit anti‐vWF (1:1000, A008229) antibodies obtained from Dako (Glostrup, Denmark), and rabbit anti‐ERG (1:3000, ab133264) and rabbit anti‐GATA3 (1:1000, ab199428) antibodies obtained from Abcam (Cambridge, MA, USA). Rabbit anti‐GAPDH (1:8000, 2118) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). A horseradish‐peroxidase (HRP)‐linked goat anti‐rabbit IgG was used as the secondary antibody. The immunoreactive bands were detected using ECL reagents (Millipore, Billerica, MA, USA), and the densitometric analysis of Western blots was performed using ImageJ software (NIH, Bethesda, MD, USA).
3
Knockdown of ERG Using siRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Knockdown of ERG was performed as previously described (40 (link)) using 1 nM siRNA oligonucleotides in OptiMEM (ThermoFisher Scientific) with Lipofectamine 2000 (ThermoFisher Scientific). Transfections were performed in serum-free media for 4 hours, then cells were grown in full growth media for 48 hours. All siRNAs and qPCR primers used in this study are listed in Table S18 . Transfection efficiency for the siRNAs utilized in this study was verified using qPCR 7 days after transfection (Figure S11A in the Data Supplement ). Protein knockdown is shown 2 days after transfection using the same siRNAs from a representative experiment (Figure S11B in the Data Supplement ). Antibodies used included 1:1,000 recombinant anti-ERG antibody (ab133264) and 1:5,000 anti-histone H3 antibody (ab1791) (Abcam).
4
Detecting ERG Protein in Leukemia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cells lysates of leukemia samples at diagnosis were obtained from cryopreserved bone marrow mononuclear cells. Anti-ERG rabbit monoclonal antibody [EPR3864(2)] (ab133264, Abcam) was used to detect ERG proteins, anti-β-actin-HRP (A3854, Sigma) was used as loading control.
5
Comprehensive Immunohistochemical Profiling of Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry was performed to assess the expression of AR (rabbit polyclonal, 22089‐1‐AP, Proteintech), ERG (rabbit monoclonal, ab133264, Abcam), PTEN (rabbit polyclonal, 22034‐1‐Ig, Proteintech), p‐AKT (mouse monoclonal, 66644‐1‐Ig, Proteintech), Beclin‐1 (rabbit polyclonal, 11306‐1‐Ig, Proteintech), Bcl‐2 (rabbit polyclonal, 12789‐1‐AP, Proteintech), Ki‐67 (rabbit polyclonal, 27309‐1‐AP, Proteintech), CD3 (rabbit polyclonal, 17617‐1‐AP, Proteintech), CD4 (rabbit polyclonal, 19068‐1‐AP, Proteintech), CD8 (rabbit polyclonal, ab4055, Abcam), IFN‐γ (rabbit polyclonal, 18013‐1‐AP, Proteintech), and TNF‐α (rabbit polyclonal, 17590‐1‐AP, Proteintech).
The expression of AR, ERG, PTEN, p‐AKT, Bcl‐2, Beclin‐1, IFN‐γ, and TNF‐α was quantified using the Pannoramic viewer and Quant center image analysis software (3D HISTECH, Hungary) to calculate the H‐score. The H‐score was calculated as follows: percent of weak staining (scale: 0‐100)*1 + percent of moderate staining (scale: 0‐100)*2 + percent of strong staining (scale: 0‐100)*3. For Ki‐67, the results were represented as the ratio of positive cells to total cancer cells. The density of tissue infiltrating lymphocytes (CD3+, CD4+ and CD8+) was calculated as the counts of CD3‐, CD4‐ and CD8‐positive cells per mm2. Representative staining specimens are shown in Figure1 .
The expression of AR, ERG, PTEN, p‐AKT, Bcl‐2, Beclin‐1, IFN‐γ, and TNF‐α was quantified using the Pannoramic viewer and Quant center image analysis software (3D HISTECH, Hungary) to calculate the H‐score. The H‐score was calculated as follows: percent of weak staining (scale: 0‐100)*1 + percent of moderate staining (scale: 0‐100)*2 + percent of strong staining (scale: 0‐100)*3. For Ki‐67, the results were represented as the ratio of positive cells to total cancer cells. The density of tissue infiltrating lymphocytes (CD3+, CD4+ and CD8+) was calculated as the counts of CD3‐, CD4‐ and CD8‐positive cells per mm2. Representative staining specimens are shown in Figure
6
Immunoblotting Analysis of Endothelial Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell protein lysates were prepared from HUVEC using CelLytic reagent (Sigma). Immunoblotting of cell lysates was performed according to standard conditions. Immunoblots were labelled with the following primary antibodies: anti-Akt (11E7) (4685, 1:1,000, Cell Signaling Technology), anti-phospho (S473)-Akt (9271, 1:1,000, Cell Signaling Technology), anti-ERG (sc353, 1:500, Santa Cruz Biotechnology), anti-ERG (ab133264, 1:1,000, Abcam), anti-Dll4 (1:500, R&D systems), anti-GAPDH (MAB374, 1:10,000, Millipore), anti-Jag1 (sc-6011, 1:1,000, Santa Cruz Biotechnology), anti-NICD/cleaved Notch1 (Val1744) (2421, 1:500, Cell Signaling), anti-Tie2 (D9D10) (7473, 1:1,000, Cell Signaling). Primary antibodies were detected using fluorescently labelled secondary antibodies: goat anti-rabbit IgG DyLight 680 and goat anti-mouse IgG Dylight 800 (Thermo Scientific). Detection and quantification of fluorescence intensity were performed using an Odyssey CLx imaging system (LI-COR Biosciences, Lincoln) and Odyssey 2.1 software. In some instances, HRP-conjugated secondary antibodies were used for chemiluminescence detection and protein levels were quantified by densitometry and normalized against loading controls. See Supplementary Fig. 10 for the uncropped immunoblots.
7
Immunoprecipitation and Western Blotting Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies for immunoprecipitation were anti-LSD1 (ab17721, Abcam, Cambridge, UK), anti-GFI1B (sc-28356X, Santa Cruz Biotech, Dallas, TX, USA), anti-RUNX1 (ab23980, Abcam), anti-H3K27ac (39133, Active Motif, Carlsbad, CA, USA), anti-CoREST (ab32631, Abcam), normal rabbit IgG (sc-2027, Santa Cruz), and normal mouse IgG (sc-2025, Santa Cruz). The primary antibodies for western blotting were anti-LSD1 (C69G12, Millipore, Billerica, MA, USA), anti-CoREST (ab32631, Abcam), anti-HDAC1 (ab7028, Abcam), anti-HDAC2 (ab7029, Abcam), anti-GFI1B (sc-28356X or sc-22795, Santa Cruz), anti-RUNX1 (ab23980, Abcam, or sc-365644, Santa Cruz), anti-ERG (ab133264, Abcam), and anti-ACTIN (sc-1616, Santa Cruz). The secondary antibodies were anti-rabbit IgG (NA934V, GE Healthcare, Little Chalfont, UK), anti-mouse IgG (NA931V, GE Healthcare), and anti-goat IgG (sc-2020, Santa Cruz) conjugated with horseradish peroxidase.
8
Nuclear Protein Extraction and EMSA Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear protein extracts were prepared from HRGECs using Pierce NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following the manufacturer's protocol. Electrophoretic mobility shift assay was performed using a LightShift Chemiluminescent electrophoretic mobility shift assay Kit (Thermo Fisher Scientific). The probes used were as follows: 5′-CAATAACAGGAAACCATCCCAGGGGGAAGTAAACCAG-3′ (probe 1); 5′-GGTGATGACACCTGCCTGTAGCATTCCAA-3′ (probe 2). Equal amounts of nuclear extract were incubated with the biotin-labeled double-stranded probes or control poly (dI:dC) for 20 minutes in binding reaction buffer. Antibodies for ERG (ab133264, Abcam) and SNAI1 (AF3639, R&D systems) were used for supershift assay. The DNA-protein complexes were electrophoresed through a nondenaturing 6% polyacrylamide gel and transferred onto a positively charged nylon membrane (Thermo Fisher Scientific). The membrane was then crosslinked with UV radiation and visualized using chemiluminescence reagents (Millipore).
9
Chromatin Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HRGECs were fixed with 1% PFA for 10 minutes, washed twice with PBS containing an EDTA-free protease inhibitor mixture (Roche, Basel, Switzerland), and collected by a cell scraper. Fragmentation of genomic DNA was achieved by sonication with a Scientz Sonifier. Immunoprecipitation was performed using a chromatin immunoprecipitation assay kit (17-371; Upstate Bio-technology, Lake Placid, NY) with antibodies for ERG (ab133264, Abcam) and SNAI1 (AF3639, R&D systems) according to the manufacturer's instructions. Relative IgG (Proteintech) was used as negative control. The target genomic DNA fragment was amplified by semi-quantitative PCR (primer sequences were listed in Table II in the Data Supplement). The PCR products were separated on 1% agarose gel and visualized under UV light.
10
Immunoblotting for ERG and GAPDH detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed according to standard conditions. Proteins were labeled with the following primary antibodies: rabbit anti-human ERG antibody (1:1,000; ab133264; Abcam) and mouse anti-human GAPDH (1:10,000; MAB374; Millipore). Primary antibodies were detected using fluorescently labeled secondary antibodies: goat anti-rabbit IgG DyLight 680 and goat anti-mouse IgG Dylight 800 (Thermo Scientific). Detection of fluorescence intensity was performed using an Odyssey CLx imaging system (Li-COR Biosciences, Lincoln) and Odyssey v.4 software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!