The 3′ end of purified RNA was biotinylated with a biotin RNA labeling mixture (Ambion, Austin, TX, USA). Next, 1 μg of labeled RNA was heated in RNA structure buffer [10 mmol/L Tris (pH 7), 0.1 mol/L KCl, and 10 mmol/L MgCl2] at 95 °C for 2 min, incubated on ice for 3 min, and incubated at room temperature for 30 min to allow the RNA to form a suitable secondary structure. Next, 3 μg of HCC stem cells were lysed with cell lysis buffer (Sigma, St. Louis, MO, USA) at 4 °C for 1 h and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatant was collected and transferred to an RNase-free centrifuge tube. Subsequently, 400 ng of biotinylated RNA was mixed with 500 μL of RIP buffer and incubated with the cell lysate mixture for 1 h at room temperature. Streptavidin-coated magnetic beads were added to each binding reaction system and incubated at room temperature for 1 h. Finally, the cells were washed five times with RIP buffer and incubated with 5× loading buffer for 5 min at 95 °C. Western blot analysis was performed to measure the amount of eluted DNMT1 protein, with rabbit anti-DNMT1 (ab183403, 1:1000, Abcam, Cambridge, MA, USA) used as the primary antibody.
Ab183403
Manufactured by Abcam
Sourced in United States
Ab183403 is a recombinant polyclonal antibody. It is produced in Escherichia coli and purified by protein A affinity chromatography.
Automatically generated - may contain errors
5 protocols using ab183403
1
RNA-Protein Interaction Profiling
2
HOXA11-AS Binds to DNMT1 in Cells
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The binding of HOXA11-AS to DNMT1 was detected by using a RNA-binding protein immunoprecipitation (RIP) Kit (Millipore, Bedford, MA, USA)22 (link). In brief, cells were lysed, and the lysate was collected. A portion of the lysate was used as input, and the other part was incubated with antibodies and magnetic beads for binding. The magnetic bead–antibody complexes were washed and resuspended in 900 μL of RIP Wash Buffer. The samples were placed on a magnetic stand to collect the magnetic bead–protein complexes for RNA extraction. The antibodies used in the RIP assay were as follows: anti-DNMT1 (ab183403; 1:50, Abcam, Cambridge, MA, USA), which was mixed at room temperature for 30 min, and IgG (ab109489, 1:100, Abcam, Cambridge, MA, USA), which served as the NC23 .
3
ChIP-qPCR for DNMT1 binding
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Cells were fixed with 1% formaldehyde at room temperature for 10 min to crosslink DNA and proteins. After crosslinking, the crosslinked DNA and proteins were randomly fragmented by ultrasonic treatment, and the supernatant was collected into three tubes. Next, anti-RNA polymerase II antibody (positive control), normal mouse IgG (NC) (ab109489, 1:100, Abcam, Cambridge, MA, USA), and an antibody specific for the target protein mouse anti-DNMT1 (ab183403, 1:50, Abcam, Cambridge, MA, USA) were added to the cells in the three tubes and incubated overnight at 4 °C. On the following day, the endogenous DNA–protein complexes were precipitated by using Protein A/G-Sepharose and briefly centrifuged to discard the supernatant. The nonspecific complexes were washed, decrosslinked at 65 °C overnight, and purified by phenol/chloroform to extract the DNA fragments. Finally, the binding of DNMT1 to the HOXA11 promoter region was detected by using primers specific for the HOXA11 gene promoter region21 (link).
4
Enrichment of DNMT1-ADAM10 Promoter Complex
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After treating with 4% formaldehyde (final concentration of formaldehyde was 1%), the collected cells were sonicated, added with the mouse monoclonal antibody to DNMT1 (ab183403, 1:50, Abcam Inc., Cambridge, MA, USA), and bound to the DNMT1-ADAM10 promoter. Protein A Agarose/SaLmon Sperm DNA were then added to the cells, bound to the DNMT1 antibody-DNMT1-ADAM10 promoter complex. The precipitated complex was washed in order to remove some non-specific binding, and eluted to obtain the enriched DNMT1-ADAM10 promoter complex which was decross-linked. The enriched ADAM10 promoter fragment was purified and subjected to PCR analysis, and IgG (ab109489, 1:100, Abcam Inc., Cambridge, MA, USA) was used as a NC [48 (link)].
5
RIP Assay for CDKN2B-AS1 and DNMT1 Interaction
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The binding of CDKN2B-AS1 to DNMT1 protein was detected using a RIP kit (Millipore, Bedford, MA, USA). The cells to be examined and washed with pre-cooled PBS, after which the supernatant was discarded. The cells were lysed with an equal volume of RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Shanghai, China) on an ice bath for 5 min, and centrifuged at 35068 ×g for 10 min at 4°C. The cell extract was co-precipitated by incubation means with the antibody. The specific steps employed were as follows: 50 μL of magnetic beads in each co-precipitation reaction system was washed, resuspended in 100 μL of RIP wash buffer, followed by the addition of 5 μg of antibody for binding based on grouping. The magnetic bead-antibody complex was washed and resuspended in 900 μL of RIP wash buffer and incubated with 100 μL of cell extraction overnight at 4°C. The sample was placed on a magnetic stand to collect the magnetic bead-protein complex. The sample was detached with proteinase K to extract RNA for subsequent PCR detection. The antibody used for RIP was DNMT1 (ab183403, 1:50, Abcam Inc., Cambridge, MA, USA) which was mixed with the complex at room temperature for 30 min, with IgG (ab109489, 1:100, Abcam Inc., Cambridge, MA, USA) as a NC [49 (link)].
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