The interactions between HNRNPA1 and miR-483-5p, HNRNPA2/B1, and miR-483-5p were assessed using an RNA immunoprecipitation kit (Geneseed Biotech). Specifically, the primary mouse renal TECs were lysed and then incubated with an RIP buffer containing anti-HNRNPA1 (Abcam) and anti-HNRNPA2/B1 (Abcam) conjugated magnetic beads. The expression of miR-483-5p was tested by qRT-PCR.
Anti hnrnpa2b1
Manufactured by Abcam
Sourced in Hong Kong
Anti-hnRNPA2B1 is a primary antibody that recognizes the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2B1) protein. hnRNPA2B1 is a RNA-binding protein involved in various cellular processes, including mRNA processing and transport.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Anti purb, by Abcam (2 mentions)
Anti igg, by Abnova (2 mentions)
Anti β tubulin, by Merck Group (2 mentions)
Anti hnrnpa1, by Abcam (1 mentions)
Rna immunoprecipitation kit, by Geneseed (1 mentions)
Hrp linked anti mouse igg, by Cell Signaling Technology (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
D 001810 01 20, by Horizon Discovery (1 mentions)
Alexa fluor 488 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Universal probe library, by Roche (1 mentions)
Anti β catenin, by BD (1 mentions)
Si hnrnpa2b1, by RiboBio (1 mentions)
Anti tubulin, by Abcam (1 mentions)
Anti histone, by Abcam (1 mentions)
Lipofectaminetm 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by ABclonal (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Anti actin, by MP Biomedicals (1 mentions)
Anti hnrnpd, by Merck Group (1 mentions)
13 protocols using anti hnrnpa2b1
1
Mapping HNRNPA1 and HNRNPA2/B1 Interactions
2
Quantitative analysis of hnRNPA2B1 and β-catenin
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The following antibodies were used: anti-hnRNPA2B1 (Abcam; DP3B3), anti-α-tubulin (TU-02, Santa Cruz Biotechnology) anti-β-catenin (C19220, BD Biosciences), anti-HA (haemagglutinin)-Tag (6E2, Cell Signaling), anti-mouse IgG HRP-linked (7076, Cell Signaling), Alexa Fluor® 488 anti-Mouse IgG (A-11001, Invitrogen). The following plasmids have been described previously: pcDNA3-HA-hnRNPA2- cDNA,34 (link) pCAGPM-HA-hnRNPA2,35 (link) pMiR-Report -3′-UTR- β-catenin.23 (link) Sequences used to generate siRNA duplexes were as previously described12 (link): si1 5′-GAAUUAUUUA AUAACAUUA-3′ and si2 5′-GAAGAGUAGU UGAGCCAAA-3′, and a non-silencing control (# D-001810-01-20, Dharmacon) was also used. Sequences used to generate oligonucleotide primers for PCR were as follows: CTNNB1_F 5′-gctttcagtt gagctgacca-3′ and CTNNB1_R 5′-caagtccaag atcagcagtc tc-3′, CASC3_F 5′-ggggttccag ttaatacaag tttc-3′ and CASC3_r 5′-gccagctgta tttctcttct gag-3′, and, ACTB_F 5’-attggcaatgagcggttc-3’ and ACTB_R 5’-cgtggatgccacaggact-3’. Universal Probe Library (04683633001, Roche) short hydrolysis probes 21 (CTNNB1), 84 (CASC3), and 11 (ACTB) were used for qPCR analysis.
3
Profiling EV-71 Infection Mechanisms
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Protease inhibitor (PMSF, Beyotime, China); Cell RIPA lysate (Beyotime, China); Cell nuclear staining solution dihydrochloride (DAPI, Beyotime, China). Antibodies: anti-VP1 (gift from Zhenjiang First People's Hospital), anti-HNRNPA2B1 (Abcam, USA), anti-GAPDH (Abclonal, China), anti-histone (Abcam, USA), anti-tubulin (Abcam, USA); anti-HRP-IgG (Abcam, USA), anti-IgG H&L (Alexa Fluor® 594) fluorescent secondary antibody (Abcam, USA), Nuclear Plasma Isolation Kit (Beyotime, China), LipofectamineTM 2000 Transfection Reagent (Thermo Fisher Scientific, USA), si-HNRNPA2B1 (Ribobio, China).
Primer: EV-71: forward, 5’-GCTCTATAGGAGATAGTGTGAGTAGGG-3’, and reverse, 5’-ATGACTGCTCACCTGCGTGTT-3’. HNRNPA2B1: forward, 5’-GCTTAAGCTTTGAAACCACAGA-3’; and reverse, 5’-CTTGATCTTTTGCTTGCAGGAT-3’. The GAPDH primers were purchased from Shanghai Bioengineering Company.
Primer: EV-71: forward, 5’-GCTCTATAGGAGATAGTGTGAGTAGGG-3’, and reverse, 5’-ATGACTGCTCACCTGCGTGTT-3’. HNRNPA2B1: forward, 5’-GCTTAAGCTTTGAAACCACAGA-3’; and reverse, 5’-CTTGATCTTTTGCTTGCAGGAT-3’. The GAPDH primers were purchased from Shanghai Bioengineering Company.
4
Investigating Synaptic Protein Regulation
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Antibodies used in this study are as follows: anti-GluA1, anti–hnRNP A2/B1, anti–PSD-95 (Abcam), anti–phospho-RPS6, anti–phospho-ERK (extracellular signal–regulated kinase), anti-FMRP (Cell Signaling), anti–N-term-GluA1, anti–N-term-GluA2, anti–hnRNP D (Millipore), anti-MAP2, anti–hnRNP A1, anti–14-3-3ζ (Santa Cruz), anti–hnRNP Q, anti-Flag (Sigma-Aldrich), anti-NMDAR1 (Synaptic Systems), and anti-actin (MPBIO). To inhibit translation, SHSY5Y cells were treated with 10 nM RAD001 or cycloheximide (100 μg/ml) and harvested at the indicated times. To block cap-dependent translation or induce synaptic stimulation, neurons were treated with 20 nM rapamycin (Sigma-Aldrich) or recombinant BDNF (100 ng/ml) (PeproTech), respectively.
5
Deoxynivalenol and Bortezomib Signaling Assay
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Deoxynivalenol (catalog # D0156) and cyanogen bromide-activated sepharose (catalog # S9142-1) are from Sigma-Aldrich. Bortezomib (catalog # ab142123) is from Abcam, and oprozomib (catalog # S7049) is from SelleckChem.
siRNAs that targeted host proteins were from the OnTARGETplus series from Horizon Discovery: siGRP78 (catalog # L-1008198-01), siRPLP1 (catalog # L-011135-00), siRPLP2 (catalog # L-004314-01), siSRSF1 (catalog # L-018672-01), siHNRNPA2B1 (L-011690-01), siDNAJB9 (catalog # L-012815-00), and siDNAJB12 (catalog # L-020585-00).
The majority of primary antibodies and horseradish peroxidase-conjugated secondary antibodies were from Abcam: anti-GRP78 (catalog # ab191023), anti-RPLP1 (catalog # ab121190), anti-RPLP2 (catalog # ab154958), anti-HNRNPA2B1(catalog # ab6102), anti-β-actin (catalog # ab8227), anti-GAPDH (catalog # ab8245), goat anti-rabbit-HRP (catalog # ab6721), and goat anti-mouse-HRP (catalog # ab6789). Anti-SRSF1 is from Invitrogen (catalog # 32-4600). Anti-HBsAg is from Fitzgerald Industries (catalog # 20-HR20). Anti-HBV polymerase is from Santa Cruz Biotech (catalog # Sc-81590). Anti-ubiquitin-HRP P4D1 is from Cytoskeleton (catalog # 140495).
siRNAs that targeted host proteins were from the OnTARGETplus series from Horizon Discovery: siGRP78 (catalog # L-1008198-01), siRPLP1 (catalog # L-011135-00), siRPLP2 (catalog # L-004314-01), siSRSF1 (catalog # L-018672-01), siHNRNPA2B1 (L-011690-01), siDNAJB9 (catalog # L-012815-00), and siDNAJB12 (catalog # L-020585-00).
The majority of primary antibodies and horseradish peroxidase-conjugated secondary antibodies were from Abcam: anti-GRP78 (catalog # ab191023), anti-RPLP1 (catalog # ab121190), anti-RPLP2 (catalog # ab154958), anti-HNRNPA2B1(catalog # ab6102), anti-β-actin (catalog # ab8227), anti-GAPDH (catalog # ab8245), goat anti-rabbit-HRP (catalog # ab6721), and goat anti-mouse-HRP (catalog # ab6789). Anti-SRSF1 is from Invitrogen (catalog # 32-4600). Anti-HBsAg is from Fitzgerald Industries (catalog # 20-HR20). Anti-HBV polymerase is from Santa Cruz Biotech (catalog # Sc-81590). Anti-ubiquitin-HRP P4D1 is from Cytoskeleton (catalog # 140495).
6
Exosome Protein Extraction and Analysis
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Exosome-derived proteins were extracted from exosomes using a Qproteome Mammalian Protein Prep Kit (Qiagen). Proteins from cell lysates were then analyzed using western blotting. Cells were first harvested and then suspended in RIPA lysis buffer (Beyotime, Haimen, China) containing 1 mM PMSF and a protease inhibitor cocktail. Protein content was quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, United States) and then separated with 10% SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, United States), blocked with 5% BSA, and then incubated overnight at 4°C with the following primary antibodies: anti-hnRNPA2B1 (1:1,000; Abcam), anti-CXCL12 (1:1,000; Bioss, Beijing, China), anti-CXCR4 (1:100, Abcam) and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Abcam) for 1 h at room temperature. Enhanced chemiluminescent (ECL) reagent (Pierce, Rockford, IL, United States) was used to visualize the signals.
7
Western Blotting Technique for Protein Analysis
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Proteins for Western blotting were extracted from cells using the radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 1% Na-DOC, 0,1% SDS, 1% Triton X-100) with 30 min of incubation on ice and occasional vortexing. Western blotting was performed as described previously (60 (link)). The following antibodies were used: anti-hnRNP A1 (04-1469; Millipore), anti-hnRNP A2B1 (ab227465; Abcam), anti-HPV16 E7 (GTX133411; GeneTex), anti-beta tubulin (T9026; Sigma-Aldrich), anti-actin (SC-1616; Santa Cruz), anti-HA tag (SC7392; Santa Cruz), and anti-GST (A5800; Invitrogen) antibody. Secondary antibodies conjugated with horseradish peroxidase were used, and proteins were detected using the Clarity Western ECL substrate (Bio-Rad) or the Super Signal West Femto chemiluminescence substrate (Pierce).
8
Comprehensive Immunoblotting Protocol
9
Protein Extraction and Western Blot Analysis
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Cell proteins were extracted using the RIPA Lysis and Extraction Buffer (Themo Fisher Scientific). Concentrations of the extracted proteins were measured with the BCA assay, and 40–60 µg of the proteins were boiled in the Laemmli Sample Buffer (Bio-Rad) at 95℃ for 5 min, followed by 10% SDS–PAGE. The gel results were transferred onto the polyvinylidene difluoride (PVDF) membranes (Millipore) which were then blocked with 5% non-fat milk for 30 min and incubated with a primary antibody, followed by a secondary antibody to generate the signals detectable by an enhanced chemiluminescence system (Pierce). Primary antibodies used for blotting included anti-VDR (#12550 from Cell Signaling Technology), anti-hnRNPA2B1 (ab6102 from Abcam), anti-β-catenin (ab6302 from Abcam), anti-p-β-catenin (ser33/37/thr41) (#9561 from Cell Signaling Technology), anti-β-catenin (#8480 from Cell Signaling Technology), anti-LEF1 (#2230 from Cell Signaling Technology), anti-TCF1/TCF7(#2203 from Cell Signaling Technology), anti-cMYC (#18583 from Cell Signaling Technology), anti-p-GSK3β(Y216) (ab75745 from Abcam), anti-GSK3β (#12456 from Cell Signaling Technology, and anti-β-actin (A2228 from Sigma-Aldrich) antibodies.
10
Western Blot Analysis of Stem Cell Markers
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Whole-cell lysate or nuclear extract was subjected to immunoblotting analysis using standard methods. Proteins were separated by 10 % SDS-PAGE and transferred onto PVDF membranes (Millipore Corporation, Billerica, MA, USA). Membranes were blocked overnight with 5 % non-fat dried milk for 2 h and incubated with anti-hnRNPA2B1 (1:1000, Abcam) anti-CD44 (1:2000, Cell Signaling Technology) or anti-Sox2 (1:1000; Cell Signaling Technology) antibody overnight at 4 °C. After washing with TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1 % Tween 20), the membranes were incubated for 2 h at room temperature with goat anti-rabbit or goat anti-mouse antibody (Zsgb-bio, China). All the experiments were repeated at least once with similar results. ImageJ software was used to quantify the Western blot results.
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