Mouse nESCs (1 × 107) were subjected to nuclei isolation as described (Doi et al., 2009 (link)). Nuclei were then resuspended in 1 mL of cold RNA immunoprecipitation (RIP) buffer (150 mM KCl, 25 mM Tris [pH 7.4], 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, 100 U/mL RNAase inhibitor, 1× Complete) and sheared by sonication. For immunoprecipitation (IP), 2 μg of specific antibody (HNRNPK [Abcam, ab39975]; PTBP1 [Invitrogen, 324800]) or the control IgG (Cell Signaling Technologies, 2,729) was incubated with 6–10 mg supernatant at 4°C for 2 hr, then 20 μL of protein G magnetic beads (Invitrogen) were added to each IP sample and incubated at 4°C for 1 hr. The beads were collected and washed three times with RIP buffer. RNAs were extracted with TRIzol, and enrichment for each gene was determined by qRT-PCR.
Hnrnp k
Manufactured by Abcam
Sourced in United States
HnRNP K is a nuclear ribonucleoprotein that binds to single-stranded DNA and RNA. It is involved in the regulation of gene expression, RNA processing, and translation.
Automatically generated - may contain errors
Lab products found in correlation
Normal mouse igg, by Santa Cruz Biotechnology (2 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (2 mentions)
Bio spin 30 columns, by Bio-Rad (2 mentions)
Bas2500 phosphoimager, by GE Healthcare (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Protein g magnetic beads, by Thermo Fisher Scientific (1 mentions)
Ptbp1, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Cdc25a, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Amersham hyperfilm, by GE Healthcare (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Gs 800, by Bio-Rad (1 mentions)
Hnrnp l, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
α synuclein, by BD (1 mentions)
9 protocols using hnrnp k
1
Identification of RNA-Protein Interactions
2
Immunoprecipitation and Western Blotting of hnRNP K
3
Protein-DNA Binding Assay Protocol
4
Western Blotting Analysis of Key Proteins
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Western blotting was performed as previously described [16 (link), 17 (link)]. Primary antibodies specific to AR (1:200, Santa Cruz, CA, USA), PSA, OPRK1, TMPRSS2, GAPDH (1:1000, Cell Signaling Technology, MA, USA), hnRNPK (1:1000, Abcam, Massachusetts, USA) were used. The blots were then incubated with goat anti-rabbit or anti-mouse secondary antibody (Cell Signaling Technology, MA, USA) and visualized using enhanced chemiluminescence.
5
Western Blot Analysis of Proteins
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Western blot was conducted as we previously described.41 (link) Briefly, total proteins were extracted from indicated NSCLC cells using RIPA lysis buffer (Beyotime, Shanghai, China). Proteins enriched from RNA pull-down assays or extracted from cells (30 μg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by being transferred into nitrocellulose membrane (Millipore). After blocking using 5% non-fat milk, the membranes were incubated with primary antibodies against hnRNPK (Abcam), GAPDH (Abcam), MYC (Cell Signaling Technology), or CDC25A (Cell Signaling Technology). The membranes were further incubated with horseradish peroxidase conjugated goat anti-rabbit or goat anti-mouse second antibodies, followed by visualization using enhanced chemiluminescence.
6
Normalized Quantitative Western Blotting
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For Western blot analysis, equal amounts of protein (adjusted from Coomassie stains) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Whatman). After blocking with 5% (w/v) low-fat milk in TBS/0,1%/Tween-20 (Roth), quantitative immunoblot analyses were performed using primary antibodies against hnRNP K (polyclonal, anti-rabbit, 1:500, Abcam), hnRNP L (monoclonal, anti-mouse, 1:2000, Abcam), α-synuclein (monoclonal, anti-mouse, 1:1000, BD Biosciences), NEFL (monoclonal, anti-rabbit, 1:50000, Novus), and β-actin (monoclonal, anti-mouse, 1:4000, Sigma), followed by appropriate HRP-coupled secondary antibodies in 5% low-fat milk/TBS/0,1% Tween-20. Immunoreactive bands were detected using HRP-chemiluminescence substrate (Pierce ECL Western Blotting Substrate, Thermo Scientific) and exposition on Amersham Hyperfilm (GE Healthcare). Signals on the developed films were digitised (GS-800, BioRad) and quantified using QuantityOne v4.6.9 (BioRad). Signals were normalised to Coomassie stains because the present proteome analyses have revealed that the abundance of proteins commonly used for normalisation (e.g., GAPDH and cytoskeletal components, such as tubulin and actin isoforms) also showed changes in response to drug treatment (cf. Additional file 1 : Table S1).
7
Protein-DNA Binding Assay Protocol
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Both duplex and single-stranded oligonucleotides were end-labeled with γ-P32 dATP using T4 polynucleotide kinase (New England Biolabs) and unincorporated nucleotides were removed using Bio-30 spin columns (Bio-Rad). All protein/DNA binding reactions were conducted in 20μL with 5mM HEPES (pH 7.9), 25mM KCl, 0.05mM EDTA, 0.125mM phenylmethysulfonyl fluoride, 1mg/mL of non-specific herring sperm carrier DNA, 0.0l% NP40 and 2μL of radiolabeled DNA probe. Binding reactions either used purified protein stocks of hnRNP K (Abcam) or CREB (generous gift from Dr. Jennifer K. Nyborg, Colorado State University). Binding reactions were carried out at 4°C for one hour before being resolved on either 3% or 5% 29:1 acrylamide/bis-acrylamide gels with 1/4x TBE buffer at 215V for two hours. Gels were dried and then imaged using a BAS2500 phosphoimager (GE Healthcare). The duplex oligonucleotides used for the EMSAs were the same as the wild-type sequences used for the protein pull-down assays and mass spectrometry analysis (see above).
8
Western Blot Analysis of Hematopoietic Proteins
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Cells were lysed in a buffer containing 100 mM Tris (pH 7.6), 1% Triton X-100, 150 mM NaCl, 0.1 mg aprotinin, 35 mg/ml PMSF 10 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, and 4 mM EDTA (approximately 5 × 106 cells). Samples were centrifuged at 4°C for 20 min to remove cell debris. Protein concentration was measured using the Bradford Assay (Bio-Rad). Laemmli buffer containing 100 mmol/L of dithiothreitol was added to the protein extracts and heated at 100°C for 5 min. Samples were run on a 10% SDS-PAGE. After the run, the proteins were transferred to nitrocellulose membranes (Millipore). Membranes were immunoblotted with NR4A3 (Abcam, ab41918), HnRNPK (Abcam, ab32969), SF3B2 (ProteinTech, 10919-1-AP), HnRNPA1 (ProteinTech, 11176-1-AP), PARP1 (Santa Cruz, sc-56197), Lamin B1 (Santa Cruz, sc-6127), and GAPDH (Santa Cruz, sc-32233) antibodies. K-562 protein samples were obtained from three independent experiments, all bands are shown in the Supplementary Material
9
Immunoprecipitation of hnRNP K Protein
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Activated whole CD4 + T cells and Jurkat cells were pelleted and frozen at -80°C. Samples were lysed in RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholate) for western blotting (WB) and IP lysis buffer for immunoprecipitation (50mM Tris, 150mM NaCl, 1% NP-40, 0.1% Triton-X 100, 0.5% Sodium Deoxycholate). 4-10 µg of protein was run for the WB and 40-50 µg of initial protein was used for IP. Input samples were loaded as 10% of IP protein loading. Samples were run on 7.5% SDS-PAGE gels and transferred onto nitrocellulose membranes. Blots were blocked with 1% milk protein in TBS-Tween(0.1%). IP was performed according to manufacturer's instructions provided by Santa Cruz Biotechnology. IP antibodies used were hnRNP K (Abcam, catalog no. -ab39975) and normal mouse IgG (Santa Cruz Biotechnology, catalog no. sc-2025). Protein A/G Plus Agarose beads were used for the pull down (Santa Cruz Biotechnology, catalog no. sc-2003). Additional information on protein isolation, western blotting, IP and blot imaging procedures can be found in Webb et al 3 (link) .
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