Antibodies to caveolin-1, phospho-AMPKα T172, AMPKα, Histone H3, eNOS, and phospho-eNOS Ser1177 were from Cell Signaling Technology (Danvers, MA). Antibodies to LKB1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa. Cruz, CA). Anti-HuR was purchased from Millipore (Temecula, CA). Anti-CD31 was from Abcam (Cambridge, UK). FuGene HD transfection reagent was from Roche Applied Science (Indianapolis, IN).
Anti hur
Manufactured by Merck Group
Anti-HuR is a laboratory research tool that binds to and inhibits the activity of the HuR protein. HuR is an RNA-binding protein involved in the regulation of gene expression. Anti-HuR can be used to study the role of HuR in various cellular processes.
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Lab products found in correlation
Phospho ampkα t172, by Cell Signaling Technology (1 mentions)
Caveolin 1, by Cell Signaling Technology (1 mentions)
Phospho enos ser1177, by Cell Signaling Technology (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Anti cd31, by Abcam (1 mentions)
Magna rip kit, by Merck Group (1 mentions)
Protein g sepharose bead, by GE Healthcare (1 mentions)
Protease phosphatase inhibitor cocktail, by Cell Signaling Technology (1 mentions)
Anti eif4g, by Cell Signaling Technology (1 mentions)
Anti ago2, by Abcam (1 mentions)
Anti yb1, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti myc tag antibody, by Cell Signaling Technology (1 mentions)
Anti peif4e ser209, by Cell Signaling Technology (1 mentions)
Anti mtor 7c10, by Cell Signaling Technology (1 mentions)
Anti gapdh 6c5, by Abcam (1 mentions)
Anti mcl 1 y37, by Abcam (1 mentions)
Anti cyclin d1 m20, by Santa Cruz Biotechnology (1 mentions)
Monoclonal anti α tubulin antibody, by Merck Group (1 mentions)
5 protocols using anti hur
1
Antibody Validation for AMPK Signaling
2
RIP Assay for RNA-Binding Proteins
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The RIP assay was performed using a Magna RIP™ kit (Millipore, Burlington, MA, USA) following the manufacturer’s protocol. Briefly, Hs578T-shSCR cells were collected and suspended in an RIP lysis buffer. Cell extracts were incubated overnight at 4 °C with magnetic beads protein A/G conjugated with 5 μg of anti-HuR (Millipore, cat. #03-102), or IgG antibody (Millipore; cat. #PP64B). Each immunoprecipitate (IP) was treated with 0.5 mg/mL proteinase K (15 min at 55 °C). RNA isolated from IP material was analyzed using RT-qPCR. GAPDH mRNA in each IP was used for normalization of RIP results. Actin (ACTB) mRNA levels were used as a positive control. The RIP assay was performed in three independent replicates assayed in triplicate.
3
Co-Immunoprecipitation of AFP and HuR
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Co-IP was performed as described previously30 (link). Briefly, cells were washed with ice-cold PBS and lysed in an immunoprecipitation assay buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA and 10% glycerol) supplemented with the Protease/Phosphatase Inhibitor Cocktail (Cell Signaling Technology). The lysates were incubated on ice for 20 min and centrifuged at 12,000 rpm for 20 min at 4 °C. The cell lysates were incubated with an anti-AFP (SC-166325, Santa Cruz) or anti-HuR (#07–468, Millipore) antibody for 4 h, and an IgG antibody was used as a negative control. Following a washing step with PBS, protein G-sepharose beads (GE healthcare) were mixed with the whole-protein lysates and incubated for 1 h at 4 °C. The beads were washed four times with the immunoprecipitation assay buffer, suspended in Laemmli buffer, and boiled for 5 min. The eluted Co-IP lysates were analyzed by Western blotting.
4
Western Blotting Antibody Detection
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Western blotting was performed as previously described [37 (link)]. The primary antibodies used were as follows: anti-Myc-tag antibody (#2276; 1:1000), anti-peIF4E Ser-209 (#9741; 1:1000), anti-Mnk1 C4C1 (#2195; 1:1000), anti-eIF4G (#2498; 1:1000), anti-YB1 (#4202; 1:1000), anti-mTOR 7C10 (#2983; 1:1000), anti-β-catenin (#9562; 1:1000; all from Cell Signaling); anti-EIF4ENIF1 (three different antibodies: 1:500; Sigma; 1:1000 Cell Signaling; 1:1000 Abnova); anti-Cyclin D1 M-20 (1:1000; Santa Cruz Biotechnology); anti-MCL1 Y37 (1:500), anti-Ago2 (1:500), anti-GAPDH 6C5 (1:2000; all from Abcam); and anti-HuR (1:500; Millipore). Anti-actin CP01 (1:500; Calbiochem, Darmstadt, Germany) was used as a loading control. The secondary antibodies used were donkey anti-rabbit IgG-HRP (NA9340; 1:2000) and donkey anti-mouse IgG-HRP (NA9340; 1:2000; both from Amersham Pharma-Biotech, Uppsala, Sweden). Bound antibodies were visualized with an enhanced chemiluminescence detection kit (Amersham Pharma-Biotech).
5
Western Blot Analysis of Protein Complexes
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Cultured cells were harvested and resuspended in Roeder D (200 mg/ml glycerol, 100 mM KCl, 0.2 mM EDTA, 100 mM Tris pH 8.0, 500 μM DTT and 200 μM PMSF). Cell lysis was carried out in a Bioruptor® Plus sonication device (Diagenode) for 10 min (low intensity settings, 30s on/off). 60 μg of proteins in cell lysates were separated on a NuPAGE™ 4-12% Bis-Tris Protein Gel (Invitrogen) and transferred onto a nitrocellulose membrane (GE) in a GENIE® blotter (Idea Scientific) at 12V for 1 h. The membrane was blocked with 1:10 Western Blocking Reagent (Roche) in TBST (20 mM Tris pH 7.5, 137 mM NaCl and 0.1% (v/v) Tween 20). Proteins were detected with the following primary antibodies in TBST containing 1:20 Western Blocking Reagent, including rabbit polyclonal anti-HuR (Millipore), rabbit polyclonal DHX9 antibody (Proteintech), monoclonal anti-α-tubulin antibody (Sigma-Aldrich) and purified mouse anti-α-Synuclein (BD Biosciences). Following three washes in TBST, the membrane was incubated in the horseradish peroxidase (HRP) conjugated secondary anti-rabbit or anti-mouse IgG antibodies (Cell Signalling Technology) and developed with chemiluminescent substrate (Thermo #34580). Quantification of western blot bands were carried out in Image Studio Lite Ver 5.2.
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