HCC cells were subjected to Western blot analyses [37 (link)]. Briefly, the lysates were separated by 8% SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) and were then transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories). The membranes were blotted with primary antibodies against ELAVL1 (Cell Signaling Technology, Danvers, MA, USA), tubulin (Oncogene Science, Cambridge, MA, USA), and Flag (DYKDDDDK) tag (Wako Pure Chemical Industries, Osaka, Japan) and horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare Life Sciences, Chicago, IL, USA). The membranes were developed using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Burlington, MA, USA), and the signals were detected using ChemiDoc XRS Systems (Bio-Rad Laboratories). Band intensity was quantified using Image Lab 4.1 software (Bio-Rad Laboratories).
Elavl1
Manufactured by Cell Signaling Technology
Sourced in United States
ELAVL1 is a protein that binds to the 3' untranslated region (3'UTR) of target mRNAs, regulating their stability and translation. It is involved in the post-transcriptional regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Ez magna rip kit, by Merck Group (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Flag dykddddk tag, by Fujifilm (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P gsk 3β, by ABclonal (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
5 protocols using elavl1
1
ELAVL1 Protein Expression Analysis
2
ELAVL1 RNA Binding Protein Immunoprecipitation
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RNA binding protein immunoprecipitation (RIP) was performed using the EZ-Magna RIP Kit (Millipore 17-700) according to the manufacturer’s protocol, and the antibody used in this assay was ELAVL1 (Cell Signaling Technology, #12582S). The primers used in this assay are listed in Supplementary 1
3
Protein Extraction and Western Blot Analysis
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Total proteins from cells were extracted using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. Primary antibodies against YWHAZ (ABclonal, #A13370), GAPDH (Proteintech, #60004-1-Ig), AKT (Cell Signaling Technology, #4691S), p-AKT (Cell Signaling Technology, #13038S), GSK-3β (Cell Signaling Technology, #9315S), p-GSK-3β (ABclonal, #AP1088), and ELAVL1 (Cell Signaling Technology, #12582S) were used in this study.
4
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) was used to extract total protein from the cells. Protein concentration was measured using a BCA Kit (Beyotime Biotechnology). The equivalent volumes of protein were subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After blocked by 5% non-fat milk, the membrane was treated overnight with primary antibodies, including those against ELAVL1 (1:1000, Cell Signaling Technology, Danvers, MA, USA) and GAPDH (1:5000; Abcam, Cambridge, MA, USA) at 4 °C. Next, the membrane was treated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000; Abcam) at room temperature for 2 h. Immunoreactive bands were measured using an enhanced chemiluminescence kit.
5
RNA Immunoprecipitation Assay Protocol
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RIP assays were performed according to the manufacturer's protocol in the EZ‐Magna RIP kit (Millipore). Briefly, cells were lysed in lysis buffer containing a protease and RNase inhibitor cocktail. The antibody used in this assay was ELAVL1 (Cell Signaling Technology, no. 12582S). Magnetic beads were preincubated with an anti‐flag antibody or anti‐rabbit IgG for 30 min at room temperature, and lysates were immunoprecipitated with beads at 4°C overnight. RNA was purified from RNA‐protein complexes tied to the beads and was then analyzed using qRT‐PCR. The primers used in this assay are listed in Table S1 .
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