ChIP assay was performed using a ChIP kit (Millipore). After reaching 70%‐80% confluence, cells collected from each group were added with 1% formaldehyde and fixed at room temperature for 10 minutes to induce DNA‐protein cross‐linking. Then, the cells were subjected to ultrasonic treatment to produce fragments of appropriate size. The fragments were centrifuged at 6540 g at 4°C with the supernatant collected into three tubes which were added with positive control antibody to RNA polymerase II, as well as NC antibodies to human immunoglobulin G and KDM5B (ab181089; Abcam) or H3K4me3 (ab12209; Abcam) for incubation at 4°C overnight. Subsequently, Protein Agarose/Sepharose was used to precipitate the endogenous DNA‐protein complex, with the supernatant discarded after centrifugation. The non‐specific complexes were washed, and de‐cross‐linking was performed following incubation at 65°C overnight. Afterwards, phenol/chloroform was added to purify and recover DNA fragments. Finally, RT‐qPCR was performed to examine the expression of miR‐448.
Ab181089
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab181089 is a lab equipment product offered by Abcam. It serves a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ab205718, by Abcam (2 mentions)
Ab12209, by Abcam (1 mentions)
Chip kit, by Merck Group (1 mentions)
Ab124905, by Abcam (1 mentions)
Bicinchoninic acid protein quantification kit, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab213224, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
Gtx100911, by GeneTex (1 mentions)
Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (1 mentions)
Bandscan 5.0 system, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Ab58599, by Abcam (1 mentions)
Anti p pi3k, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Ab224637, by Abcam (1 mentions)
Protease inhibitor, by Boster Bio (1 mentions)
Bicinchoninic acid protein assay kit, by Boster Bio (1 mentions)
12 protocols using ab181089
1
Chromatin Immunoprecipitation for miRNA Expression
2
Protein Expression Analysis in Esophageal Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
KYSE-150, TE-10 cells and ESCC cells-formed xenograft tumor tissues were lysed with radioimmunoprecipitation assay lysis buffer (Beyotime). Protein concentrations were assessed using the bicinchoninic acid protein quantification kit (Beyotime). Equal amounts of protein (50 μg) from each sample were separated using 10% SDS-PAGE and electrophoretically transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were closed with 5% BSA at 37°C for 60 min and treated with primary antibodies to GAPDH (1:10000, ab181602, Abcam, Cambridge, MA, USA), KDM5B (1:2000, ab181089, Abcam), PIK3C3 (1:2000, ab124905, Abcam), H3K4me3 (1:1000, ab213224, Abcam), Cyclin B1 (1:2000, GTX100911, GeneTex, Inc., Alton Pkwy Irvine, CA, USA), CDC2 (1:1000, GTX108120, GeneTex), pCDC2 (Tyr15, 1:2000, GTX128155, GeneTex), Beclin-1 (1:1000, #3495S, Cell Signaling Technologies, Beverly, MA, USA), LC-3I/II (1:1000, #12741S, CST), and Bcl-2 (1:1000, #4223S, CST) overnight at 4°C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-coupled secondary antibody (1:5000, ab205718, Abcam) for 60 min at room temperature. Protein bands were analyzed using Pierce Western Blotting ECL substrate kit (Thermo Fisher Scientific) and BandScan 5.0 system (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer on ice and total proteins were extracted and separated by SDS-PAGE gels, then electrophoretically transferred to polyvinylidene difluoride membranes. Western blot analysis was performed according to standard procedures. The primary antibodies included anti-FOXP2 (1:1000, ab58599, Abcam), anti-KDM5B (1:2000, ab181089, Abcam) and anti-PI3K, anti-p-PI3K, anti-AKT, anti-p-AKT (1:1000, #4257, #4228, #9272, #9271, Cell Signaling Technology), anti-GAPDH (1:10000, 60,004–1-Ig, Proteintech Group). GAPDH on the same membrane was used as a loading control. Immunoblots were visualized by ECL chemiluminescent detection system. The blots were scanned and the integrated density value (IDV) was measured on FluorChem 2.0 software.
4
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were solubilized in radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors (Boster) and cleared cell lysate was collected by centrifugation at 4°C. Protein concentration was determined using the bicinchoninic acid protein assay kit (Boster Biological Technology, Wuhan, China). Protein electrophoresis was performed using SDS-PAGE gels (10%), and the separated proteins were transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA for 2 h at room temperature and then incubated with primary antibodies at 4°C overnight: anti-KDM5B (ab181089, Abcam, 1:2,000), SOX17 (ab224637, 1:500), and GREB1 (ab72999, 1:1,000). The next day, the membranes were washed with Tris-buffered saline with Tween (3 times, 10 min each) and incubated with HRP-conjugated secondary antibody (goat anti-Rabbit immunoglobulin G [IgG], ab205719, Abcam, 1:2,000) for 1 h at room temperature. The membranes were then developed with ECL reagent (MilliporeSigma, Burlington, MA, USA). The results were quantified using ImageJ (NIH, Bethesda, MD, USA), and the intensity values were normalized to β-actin.
5
Quantifying Protein Expression Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells from each culture group were detached by trypsin, collected, and lysed with an enhanced radioimmunoprecipitation assay lysis buffer (Wuhan Boster Biological Technology Co., Ltd.) containing a protease inhibitor. After measurement of protein concentration with a bicinchoninic acid (BCA) quantitation kit (Boster Biological Technology), samples of protein were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, and electrotransferred onto a polyvinylidene fluoride membrane. The membrane was blocked in 5% bovine serum albumin (BSA) at room temperature for 1 hour and added with diluted primary antibodies (antibody to KDM5B, ab181089; Abcam; antibody to YTHDF3, Cat # 25537‐1‐AP; Proteintech; antibody to ITGA6, Cat # 3750; CST) for incubation overnight at 4°C. The following day, the membrane was washed 3 times with Tris‐buffered saline Tween‐20, re‐probed with horseradish peroxidase (HRP)‐labelled secondary antibody of goat anti‐rabbit for 1 hour at room temperature and developed with enhanced chemiluminescence working solution (EMD Millipore). Finally, ImageJ analysis software was used to quantify the grey levels of each band in the Western blot image normalized to GAPDH.
6
Investigating TGF-β Signaling Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant mouse TGF-β protein (7666-MB) was purchased from R&D Systems (Minneapolis, MN). Angiotensin II (AngII) (CSN10313) was purchased from CSNpharm (Chicago, IL). Antibodies against α-smooth muscle actin (α-SMA) (ab5694, for immunoblot analysis), phospho-SMAD3 (SMAD family member 3) (ab52093), and KDM5B (ab181089, for immunofluorescence analysis) were obtained from Abcam (Cambridge, UK). Antibodies against collagen type I (AB765p) were obtained from Millipore (Billerica, MA). Antibodies against α-SMA (5228, for immunofluorescence and immunohistochemistry) were obtained from Sigma‒Aldrich (Darmstadt, Germany). Antibodies against collagen type III (NB600-594) were obtained from Novus Biologicals (Littleton, CO). Antibodies against phospho-SMAD2 (SMAD family member 2) (3108), SMAD2 (5339), SMAD3 (9523), phospho-ERK (extracellular signal-regulated MAP kinase) (9106), ERK (4696), phospho-JNK (c-Jun N-terminal kinase) (4668), JNK (9252), phospho-p38 (4511), p38 (9212), phospho-p65 (3033 S), p65 (8242), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174) and ATF3 (18665) were obtained from Cell Signaling Technology (Danvers, MA). Antibodies against KDM5B (A301-813A, for immunoblot analysis) were obtained from Bethyl Laboratories (Montgomery, TX).
7
Immunohistochemical Analysis of JARID1B
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin-embedded sections acquired from samples were deparaffinized and rehydrated. Then, the citric acid buffer was used for antigen retrieval. Next, after H2O2 treatment and nonspecific antigen blocking for 30 min, sections were incubated with the primary JARID1B antibody (1:100, ab181089, Abcam, Cambridge, UK). Next day, the colorimetric detection was performed using DAB (3,3'-diaminobenzidine) staining kit (Vector Laboratories, USA).
8
Immunohistochemical Analysis of KDM5B Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
91 paired, paraffin-embedded primary specimens were stained for KDM5B by immunohistochemistry (IHC), as described previously [65 (link)]. The sections were incubated with anti-KDM5B (dilution ratio, 1:1000; No. ab181089 Abcam, Cambridge, UK) at 4°C overnight followed by secondary anti-rabbit HRP-conjugated antibody. The expression changes of KDM5B were evaluated blindly by two professional pathologists. Cancer cells were counted under the microscope as previously described [65 (link)].
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The radioimmunoprecipitation assay lysis buffer (Sigma, St. Louis, MO, USA) was applied to isolate the total protein from tissue samples and cells, and the BCA assay kit was used to determine the protein concentrations. Protein samples were processed by 10% SDS-polyacrylamide gelelectrophoresis and transferred onto nitrocellulose membranes.
After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice. Lastly, protein levels were measured using the enhanced-chemiluminescence reagent (Millipore, Billerica, MA, USA) and analyzed using ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD, USA).
After the blockade with 5% defatted milk at room temperature for 2 h, membranes were incubated with the following primary antibodies: KDM5B (ab181089, 1:1000, Abcam, Cambridge, MA, USA), TRIM37 (ab264190, 1:2000, Abcam, Cambridge, MA, USA), and GAPDH (ab9485, 1:2500, Abcam, Cambridge, MA, USA) at 4 ℃ overnight, and with goat anti-rabbit immunoglobulin G (horseradish peroxidase) antibody (ab205718, 1:2000, Abcam, Cambridge, MA, USA) at room temperature for 1 h after being washed with PBS thrice. Lastly, protein levels were measured using the enhanced-chemiluminescence reagent (Millipore, Billerica, MA, USA) and analyzed using ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD, USA).
10
Chromatin Immunoprecipitation and RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in the lysis buffer (50 mM Tris-HCl, 2.5 mM EDTA, 130 mM NaCl, and 1% NP-40) containing ribonuclease and protease inhibitor (ThermoFisher Scientific, Shanghai, China). A portion of the supernatants were used as the input and the other portion of the supernatants were incubated with primary antibody against KDM5B (ab181089, Abcam, Cambridge, MA, USA) or antibody against IgG (ab172730, Abcam, Cambridge, MA, USA) at 4 ℃ overnight. Samples conjugated with antibodies were incubated with protein-A Sepharose (Sigma, St. Louis, MO, USA) at 4 ℃ for 2 h, washed with washing buffer, and incubated with protease K (Sangon Biotech Co., Ltd., Shanghai, China) for 30 min. The total RNA was extracted using the TRIzol solution (Invitrogen, Carlsbad, CA, USA) for RT-qPCR.
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!