Western blot experiments were performed according to the instruction of our previous studies (19 (link)). The information of antibodies used in this study was given as followings: DDX11 (ab230017, Abcam), E2F1 (#3742, cell signaling technology), E2F2 (#ab138515, Abcam), E2F3 (#ab50917, Abcam), EZH2 (#5246, cell signaling technology), p21 (#2947, cell signaling technology), p53 (#2524, cell signaling technology), phosphorylated Rb (#8147, cell signaling technology), cyclin E (#4136, cell signaling technology), cyclin D1 (#55506, cell signaling technology), MDM2 (#86934, cell signaling technology), β-actin (#3700, cell signaling technology).
Ab138515
Manufactured by Abcam
Sourced in United States
Ab138515 is a lab equipment product offered by Abcam. It is a device used for scientific research purposes. The core function of this product is to facilitate data collection and analysis within a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Ab50917, by Abcam (1 mentions)
Ab5823, by Abcam (1 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Ab1669, by Abcam (1 mentions)
Cell lysis solution, by Beyotime (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Citric acid, by Agilent Technologies (1 mentions)
Ab15580, by Abcam (1 mentions)
Am6000, by Leica (1 mentions)
Ab237703, by Abcam (1 mentions)
Odyssey infrared laser scanner, by LI COR (1 mentions)
Ab0035, by Abways (1 mentions)
Cy5040, by Abways (1 mentions)
Ab0054, by Abways (1 mentions)
0.22 μm pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Ab192239, by Abcam (1 mentions)
Ta150121, by OriGene (1 mentions)
8 protocols using ab138515
1
Western Blot Antibody Reference
2
Histone H4 and E2F2 Binding Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extracts were obtained from undifferentiated QCMCs according to the manufacturer’s protocol and used for EMSAs using LightShift Chemiluminescent EMSA Kit (Thermo Fisher Corp., Waltham, MA, USA) with modifications. Briefly, 200 fmol of 5′-biotin labeled histone H4 or E2F2 probes (listed in Table 1 ) was incubated with 10 μg nuclear extracts, 2 μL of 10 × binding buffer, 1 μL of poly dI·dC and 1 μL of 50% glycerol in a volume of 20 μL. For the competition assay, unlabeled or mutated probes were added to the reaction mixture for 10 min before adding the labeled histone H4 or E2F2 probes. Finally, 10 μg of histone H4 (ab5823, Abcam, Cambridge, MA, USA) or E2F2 (ab138515, Abcam) antibody was added to the reaction mixture for the super-shift assay. The DNA-protein complexes were separated on a 6% non-denaturing polyacrylamide gel and based on three independent experiments. Images were captured using the molecular imager ChemiDoc™ XRS+ system (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of E2F2 and CCR4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RASF transfected with anti-E2F2 siRNA or E2F2-expressing plasmids were homogenized with cell lysis solution (Beyotime, China) and centrifuged at 12,000 × γ for 30 min at 4oC. The supernatant was collected, and protein concentrations were determined using a BCA protein assay kit (Beyotime, China). Total protein was separated on 10% SDS-PAGE gels, transferred to PVDF membranes and blocked in 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) at pH 7.5. The membranes were incubated overnight at 4oC with rabbit polyclonal antibody against human E2F2 (ab138515, Abcam, UK), goat polyclonal antibody against human CCR4 (ab1669, Abcam, UK) or rabbit polyclonal antibody against human GAPDH (Good Here, China) at a 1 : 1000 dilution. Horseradish peroxidase-conjugated sheep anti-rabbit immunoglobulin G (IgG) secondary antibody or rabbit anti-goat IgG secondary antibody was incubated with the membrane for 1 h at room temperature in TBST. Complexes were visualized using Immobilon Western chemiluminescent HRP substrate (Millipore, USA). The expression level of E2F2 or CCR4 protein was quantified by normalizing GAPDH expression using Image J software (NIH, Bethesda, MD, USA) to compare their gray values.
4
Immunohistochemical Analysis of Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor tissues were fixed with 4% paraformaldehyde (PFA, V/V), followed by paraffin embedding. 4-μm tissue sections in paraffin were soaked in xylene for 15 min before dehydration with gradient ethanol. Each section was then soaked with citric acid (pH 6.0 DAKO) for 10 min at 95° for antigen retrieval and cooled to ambient temperature. After washing with TBST buffer for a 15-min period, the sections were incubated with 3% H2O2 for 10 min. Tissue sections were blocked using 5% bovine serum albumin (BSA) for 30 min, followed by overnight incubation using primary antibodies E2F2 (Ab138515, Abcam) and Ki67 (ab15580, Abcam) under 4°C. Color development was performed using a DAB color-rendering kit (Soleibol, Japan). The images were captured using a Leica AM6000 microscope.
5
NF-κB Signaling Pathway Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer (Solarbio, Beijing, China) was utilized to separate total proteins from cell lysates. After resolution using 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, proteins were shifted onto 0.22 μm PVDF membranes (Millipore). Following 1 h of blocking in 1× TBST buffer containing 5% nonfat dry milk, an overnight incubation of the membranes was performed at 4°C using primary antibodies to NF-κB p65 (#8242, 1:1000, CST, USA), IκBα (#4814, 1:1000, CST, USA), p-IκBα (#2859 1:1000, CST, USA), IKKβ (#8943 1:1000, CST, USA), p-IKKα/β (#2697 1:1000, CST, USA), NF-κB p50 (CY5040, 1:2000, Abways, China), RAB10 (ab237703, 1:1000, Abcam, USA), E2F2 (ab138515, 1:1000, Abcam, USA), Lamin B1 (AB0054, 1:1000, Abways, China) and β-actin (AB0035, 1:1000, Abways, China). After a triple ten-minute rinse using 1× TBST buffer, the membranes were proceeded 1 hour incubation at 37°C protected from light utilizing the corresponding DyLight 800-labeled goat anti-mouse or anti-rabbit antibodies (1:7000, abbkine, China). Finally, the blot image was recorded with the Odyssey Infrared Laser Scanner (LICOR Biosciences). Three replicates were required for each experiment. β-Actin levels were employed to normalize protein levels of the whole cell lysis and Lamin B1 for nuclear extract.
6
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedures were performed according to our previous study [47] . The primary antibodies used in western blot were as follows: anti-ACTIN (20536-1-AP, Proteintech), anti-ZFHX3 (PD010, MBL), anti-FTO (27226-1-AP, Proteintech), anti-MYC (TA150121, OriGene), anti-E2F2 (ab138515, Abcam), and anti-CDKN2C (ab192239, Abcam).
7
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer (Beyotime Biotechnology, Shanghai, China) was utilized to extract total protein, and protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China) as per instructions. The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking with the skimmed milk, the membrane was incubated using the following antibodies: E2F2 (Ab138515, Abcam), actin (ab8227, Abcam), E-cadherin (ab231303, Abcam), N-cadherin (ab76057, Abcam), vimentin (ab137321, Abcam) at 1 : 1000 dilutions at 4°C overnight. The membrane was washed with the TBST buffer and then incubated with HRP-conjugated secondary antibody under ambient temperature for 2 h. The protein bands were visualized using the ECL detection kit (Yeasen, Shanghai, China), with actin as the internal control.
8
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Radioimmunoprecipitation assay (RIPA) lysis buffers (CST, Danvers, MA, USA) containing proteinase and phosphatase inhibitors were used to lyse the transfected MHCC97-H, HCCLM3, and Hep 3B cells. After being centrifuged for 30 min, a Pierce Bicinchoninic acid (BCA) Protein detection kit (Bio-Rad Laboratories, Hercules, CA, USA) was used for assessing the concentration of the proteins. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) used to separate proteins in each sample, and the proteins were then transferred to the polyvinylidene difluoride membranes (PVDF; Bio-Rad Laboratories, Hercules, CA, USA). The membranes were disposed with 5% non-fat milk and then cultured with primary antibodies E-cadherin (#3195, 1:1,000; CST), N-cadherin (#13116, 1:1,000; CST), Vimentin (#5741, 1:1,000; CST), GAPDH (ab9485, 1/10,000; Abcam), β-catenin (#8480, 1:1,000; CST), c-Myc (#18583, 1:1,000; CST), Cyclin D1 (#55506, 1:1,000; CST), MMP-7 (#71031, 1:1,000; CST), β-actin (#4970, 1:1,000; CST), E2F2 (ab138515, 1/1,000; Abcam), and SOX12 (cat. no. 23939-1-AP, 1:500; Proteintech) at 4°C overnight. Later, membranes were cultured with secondary antibodies (ab205718, 1/10,000; Abcam) at the room temperature for 1 h. The bands were visualized via enhanced chemiluminescence (ECL; Millipore, USA). GAPDH or β-actin was employed as the internal control. Experiments were conducted three times.
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!