RIP assays were performed in indicated melanoma cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) and an antibody against NF90 (5 µg per reaction; ab131004, Abcam) in accordance with the manufacturer’s manual.
Ab131004
Manufactured by Abcam
Sourced in United Kingdom, Hong Kong
Ab131004 is a recombinant monoclonal antibody targeting the human GAPDH protein. It is suitable for use in various immunoassay applications, such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Ab8245, by Abcam (2 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Ab207434, by Abcam (1 mentions)
Ab178945, by Abcam (1 mentions)
Pa5 29645, by Thermo Fisher Scientific (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Ab179445, by Abcam (1 mentions)
Ab199728, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Anti e2f1, by Abcam (1 mentions)
Map2k6, by Abcam (1 mentions)
Anti cdk6 3136s, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
Ab154291, by Abcam (1 mentions)
Phospho mek1 2, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
7 protocols using ab131004
1
NF90 RNA-Binding Protein Assay
2
Western Blot Analysis of Protein Targets
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Protein lysates were prepared using RIPA buffer (Pierce, Rockford, IL, USA), and separated by gel electrophoresis. Proteins were then transferred onto PVDF membrane (Pierce). After blocking, the blots were incubated with appropriate primary and secondary antibodies. The protein bands were detected suing ECL reagents (Pierce). The intensities of bands were quantified using Image J software (NIH) and normalized with GAPDH. Primary antibodies used in this study: anti-ATF3 (1:1000, ab207434, Abcam, Cambridge, UK), anti-ILF3 (1:2000, ab131004, Abcam), anti-iNOS (1:1000, ab178945, Abcam), anti-Arg-1 (1:5000, PA529645, Invitrogen) and anti-GAPDH (1:2000, ab8245, Abcam) antibody.
3
RNA Immunoprecipitation of NEAT1
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RIP was conducted using Magna RIP Kit (Millipore, Billerica, MA, USA). In brief, cells were lysed with RIP lysis buffer. Anti-ILF3 (2 μg, ab131004, Abcam) or normal rabbit IgG-conjugated beads were then incubated with cell lysates. qRT-PCR was employed to detect immunoprecipitated NEAT1. LincIN served as a positive control.
4
Quantitative Protein Expression Analysis
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Cell protein lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, CA, USA). GAPDH antibody was used as a control. Anti-E2F1 (ab179445, 1:1000), -CDK4 (ab199728, 1:1000), -CDK6 (ab199728, 1:1000), -Ki67 (ab16667, 1:1000), -Bcl-2 (ab182858, 1:1000), -ILF3 (ab131004, 1:1000), -MAP2K6 (ab33866, 1:1000), and -GAPDH (ab9485, 1:2000) antibodies were purchased from Abcam. Anti -CDK6 (3136s, 1:1000), -P21 (2947s, 1:1000) were purchased from Cell Signaling Technology.
5
Western Blot Antibody Optimization Protocol
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Western blot was conducted as previously described (38 (link)) with the primary antibodies: for NF90, ab131004, 1:2,000, Abcam; for EZH2, #07-689, 1:2,000, Millipore; for NRAS, ab154291, 1:1,000, Abcam; for GAPDH, ab8245, 1:5,000, Abcam; for phospho-MEK1/2, #9154, 1:1,000, Cell Signaling Technology; for MEK1/2, #8727, 1:1,000, Cell Signaling Technology; for phospho-ERK1/2, #4370, 1:2,000, Cell Signaling Technology; for ERK1/2, #4695, 1:1,000, Cell Signaling Technology.
6
Western Blot Analysis of NF90 Protein
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Total proteins were extracted from indicated ESCC cells with RIPA buffer (Beyotime, Jiangsu, China) added with protease inhibitors (Beyotime). Protein concentrations were determined by BCA assay using the BCA Protein Assay Kit (Beyotime). Equal amounts of proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by being transferred to nitrocellulose membrane (Millipore, Bedford, MA, USA). After being blocked, the membranes were incubated with NF90 specific primary antibody (ab131004, Abcam, Hong Kong, China) and β-actin specific primary antibody (66009-1-Ig, Proteintech, Rosemont, IL, USA). After three washes, the membranes were incubated with IRDye 700CW goat anti-mouse IgG (Li-Cor, Lincoln, NE, USA) and IRDye 800CW goat anti-rabbit IgG (Li-Cor), followed by being scanned on an Odyssey infrared scanner (Li-Cor).
7
RIP Assay of NF90 Interactome
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RIP assays were carried out in the TE-1 and Eca-109 cells using the EZ-Magna RIP™ RNA Binding Protein Immunoprecipitation kit (EMD Millipore) and primary antibody against NF90 (5 µg, ab131004, Abcam). The enriched RNA (LincIN and pri-miR-7) was quantified by RT-qPCR as described above.
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