Human breast tumor tissue microarrays containing 139 analyzable cases of breast carcinoma were purchased from US Biolab (#HBreD139Su01). Tissue specimens were processed as described in our previous publication5 (link). The primary antibodies used for immunohistochemical analysis were antibodies against DGCR8 (1:250, Abcam, #ab90579) and USP51 (1:250, Abcam, #ab121147). Whole-slide images were captured using an Aperio CS2 slide scanner system (Leica Biosystems, Wetzlar, Germany) at a 40× magnification. Immunohistochemical staining was analyzed by using the immunoreactive score (IRS) system. The percentage of positive cells was scored on a scale of 0–4: 0 if 0% of tumor cells were positive, 1 if 1–10% of cells were positive, 2 if 11–50% were positive, 3 if 51–80% were positive, and 4 if 81–100% were positive. Staining intensity was scored on a scale of 0–3 (3 is the strongest). Final IRS score = (score of the staining intensity) × (score of the percentage of positive cells). Score = 0–6 was considered moderate or weak expression and score = 8–12 was considered strong expression.
Ab90579
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Ab90579 is a lab equipment product. It is a tool used for scientific research purposes.
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Lab products found in correlation
Ab12286, by Abcam (3 mentions)
Ab121147, by Abcam (2 mentions)
Aperio cs2, by Leica (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions)
Ab191875, by Abcam (1 mentions)
Ab252562, by Abcam (1 mentions)
Ab223090, by Abcam (1 mentions)
Sc 2027, by Santa Cruz Biotechnology (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
T6557, by Merck Group (1 mentions)
Magna merip m6a kit, by Merck Group (1 mentions)
M6a antibody, by Merck Group (1 mentions)
Ab246514, by Abcam (1 mentions)
Ab208577, by Abcam (1 mentions)
Ab195352, by Abcam (1 mentions)
Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)
Rabbit igg hrp conjugated whole antibody, by GE Healthcare (1 mentions)
Ecl mouse igg hrp conjugated whole antibody, by GE Healthcare (1 mentions)
Anti lin28b, by Cell Signaling Technology (1 mentions)
10 protocols using ab90579
1
Breast Tumor Tissue Microarray Analysis
2
Immunoprecipitation and Western Blot Analysis
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Cell lysates were prepared with the Radioimmunoprecipitation assay (RIPA) lysis buffer. Antibodies, including anti-METTL14 (ab252562; Abcam, Cambridge, MA, United States), anti-DGCR8 (ab90579; Abcam) or normal IgG antibody (sc-2027; Santa Cruz Biotechnology, Inc.), were incubated with the cell lysates followed by incubation with Protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz Biotechnology, Inc.) for 2 h at 4°C. After washing with the lysis buffer 3 times, the samples were subjected to Western blot analysis using anti-METTL14 (ab223090; Abcam) and anti-DGCR8 (ab191875; Abcam) antibodies.
3
Profiling ADAR1, DROSHA, and DGCR8 Interactions
4
Profiling RNA processing machinery
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Primary antibodies used were directed against Drosha (ab12286, Abcam), DGCR8 (ab90579, Abcam), DBR1 (sc-99369, Santa Cruz), snRNP70 (sc-9571 c-18, Santa Cruz), PRP8 (sc-55534 F-6, Santa Cruz), U2AF65 (sc-48804 H-300, Santa Cruz), Dicer (sc-56651), Ago2 (sc-32877 H-300, Santa Cruz) and γ-Tubulin (T6557, Sigma).
5
RNA Immunoprecipitation and m6A Binding Assays
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An RNA immunoprecipitation (RIP) assay was performed with a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocol. Cells were lysed in RIP lysis buffer, and then 100 μL whole-cell extract was incubated with magnetic beads conjugated with anti-DGCR8 (ab90579; Abcam, UK) or immunoglobulin G (IgG) for 6 h at 4°C. After that, the beads were incubated with Proteinase K with shaking to remove protein. Finally, the coprecipitated RNAs were extracted and subjected to qRT-PCR using primers for primiRNAs and normalized to input.
For the m6A RNA binding assays, the Magna MeRIP m6A Kit (Millipore, Billerica, MA, USA) was used. In brief, RNAs were chemically fragmented to ∼100 nt, and fragmented RNA was then incubated with magnetic beads conjugated with m6A antibody (Millipore, Billerica, MA, USA) for immunoprecipitation. The enrichment of m6A-containing mRNA was then analyzed through qRT-PCR and normalized to input.
For the m6A RNA binding assays, the Magna MeRIP m6A Kit (Millipore, Billerica, MA, USA) was used. In brief, RNAs were chemically fragmented to ∼100 nt, and fragmented RNA was then incubated with magnetic beads conjugated with m6A antibody (Millipore, Billerica, MA, USA) for immunoprecipitation. The enrichment of m6A-containing mRNA was then analyzed through qRT-PCR and normalized to input.
6
m6A-RIP Protocol for Protein-RNA Interactions
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RIP analysis was performed with the Magna RIP RNA-binding protein immunoprecipitation kit as per the manufacturer’s protocol (Millipore, Bedford, MA). Briefly, samples were irradiated at 254 nm, 400 mJ/cm2 (Stratagene Stratalinker), followed by treatment with RIP lysis buffer. The immunoprecipitation was implemented using the with antibodies against m6A (1:800, ab208577, Abcam, Cambridge, MA), DGCR8 (1:800, ab90579, Abcam, USA), and YTHDF2 (1:1000, cat. no. ab246514, Abcam, USA) proteins. The enriched RNA was analyzed via qRT-PCR.
7
Co-immunoprecipitation of METTL3 and DGCR8
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To verify the direct binding between METTL3 and DGCR8, RN-sc cells were lysed and incubated with antibodies against METTL3 (ab195352, Abcam, USA) at 4 °C overnight. Subsequently, cell lysates were cultivated with protein A/G agarose beads (Santa Cruz, CA, USA) for 2 h. Finally, western blotting with antibodies against DGCR8 (ab90579, Abcam) were employed to analyze the respective protein expression in immuno-complexes.
8
Profiling ADAR1, DROSHA, and DGCR8 Interactions
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Ten million HeLa cells were lysed by 1 mL non-denaturing lysis buffer (20 mM Tris-HCl pH 8, 137 mM NaCl, 1% Nonidet P-40, 2 mM EDTA) with complete protease inhibitor cocktail. Co-immunoprecipitation (Co-IP) experiments were performed using 10 μg ADAR1 antibody (D-8, Santa Crutz, sc-271854), 10 μg DROSHA antibody (Abcam, ab12286), or 2 μg DGCR8 antibody (Abcam, ab90579), or corresponding isotype IgG with Dynabeads Protein G (Life Technology) at 4 °C overnight. Then Protein G-antibody-antigen complex was washed by wash buffer (10mM Tris, pH 7.4, 1mM EDTA, 1mM EGTA, pH 8.0, 150mM NaCl, 1% Triton X-100) with complete protease inhibitor cocktail. Protein complex was finally eluted from the Dynabeads using elute buffer (0.2 M glycine, pH 2.8). IP was validated by immunoblot (IB) using ADAR1 antibody (15.8.6, Santa Crutz, sc-73408, 1:1000 dilution), DROSHA antibody (Abcam, ab12286, 1:500 dilution) and DGCR8 antibody (Abcam, ab90579, 1:1000 dilution) to immunoblot the corresponding antigens. RNase A was used to degrade single stranded RNA at 20μg mL−1 for 1 h at 4°C during antigen-antibody incubation. See Supplementary Fig. 13 for uncropped immunoblot images.
9
Western Blot Analysis of Protein Samples
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Total protein samples were extracted by radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris•HCl (pH 8.0), 150 mM NaCl, 0.5% deoxycholate (DOC), 0.1% SDS, 1% Nonidet P-40]. Proteins in the cell lysates were separated by SDS/PAGE followed by semidry transfer to a PVDF membrane. Membranes were blocked for 60 min with Blocking One (Nacalai Tesque), incubated with an anti-Flag (M185-3L, MBL), anti-DGCR-8 (ab90579, abcam), anti-Lin28B (11965S, Cell signaling technologies) or antiβ-actin (A5316; Sigma Aldrich) at 4 °C overnight, rinsed, and then incubated for 1 h . CC-BY-NC-ND 4.0 International license granted bioRxiv a license to display the preprint in perpetuity. It is made available under a
The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has this version posted March 6, 2020. ; https://doi.org/10.1101/2020.02.16.951954 doi: bioRxiv preprint with ECL mouse IgG HRP-conjugated whole antibody (GE Healthcare) or rabbit IgG HRP-conjugated whole antibody (GE Healthcare). The blot was developed with the ECL Select Western Blotting Detection Reagent (GE Healthcare). The protein levels are normalized to β-actin levels in each sample.
The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has this version posted March 6, 2020. ; https://doi.org/10.1101/2020.02.16.951954 doi: bioRxiv preprint with ECL mouse IgG HRP-conjugated whole antibody (GE Healthcare) or rabbit IgG HRP-conjugated whole antibody (GE Healthcare). The blot was developed with the ECL Select Western Blotting Detection Reagent (GE Healthcare). The protein levels are normalized to β-actin levels in each sample.
10
Breast Carcinoma Tissue Microarray Analysis
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Human breast tumor tissue microarrays containing 139 analyzable cases of breast carcinoma were purchased from US Biolab (#HBreD139Su01). Tissue specimens were processed as described in our previous publication 5 . The primary antibodies used for immunohistochemical analysis were antibodies against DGCR8 (1:250, Abcam, #ab90579) and USP51 (1:250, Abcam, #ab121147).
Whole-slide images were captured using an Aperio CS2 slide scanner system (Leica Biosystems, Wetzlar, Germany) at a 40 magnification. Immunohistochemical staining was analyzed by using the immunoreactive score (IRS) system. The percentage of positive cells was scored on a scale of 0 to 4: 0 if 0% of tumor cells were positive, 1 if 1%-10% of cells were positive, 2 if 11%-50%
were positive, 3 if 51%-80% were positive, and 4 if 81%-100% were positive. Staining intensity was scored on a scale of 0 to 3 (3 is the strongest). Final IRS score = (score of the staining intensity) × (score of the percentage of positive cells). Score = 0-6 was considered moderate or weak expression and score = 8-12 was considered strong expression.
Whole-slide images were captured using an Aperio CS2 slide scanner system (Leica Biosystems, Wetzlar, Germany) at a 40 magnification. Immunohistochemical staining was analyzed by using the immunoreactive score (IRS) system. The percentage of positive cells was scored on a scale of 0 to 4: 0 if 0% of tumor cells were positive, 1 if 1%-10% of cells were positive, 2 if 11%-50%
were positive, 3 if 51%-80% were positive, and 4 if 81%-100% were positive. Staining intensity was scored on a scale of 0 to 3 (3 is the strongest). Final IRS score = (score of the staining intensity) × (score of the percentage of positive cells). Score = 0-6 was considered moderate or weak expression and score = 8-12 was considered strong expression.
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