Total proteins of cells and tissues were extracted by lysing in radioimmunoprecipitation assay buffer (RIPA buffer; Beyotime Institute of Biotechnology, Jiangsu, China) on ice for 30 min and then centrifuged at 17,000 × g for 40 min at 4°C. The concentration of protein was determined by a bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology). An equal amount of protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Tween 20 with Tris-buffered saline (TTBS) with 10% non-fat milk was used to block the membranes at room temperature for 2 h. Then, membranes were incubated in primary antibody of EIF4A3 (1:500, Proteintech, Rosemont, IL, USA), EGR3 (1:300, Santa Cruz Biotechnology, Santa Cruz, TX, USA), PKP2 (1:200, Santa Cruz Biotechnology, Santa Cruz, TX, USA), PI3K, Akt (1:500, Proteintech, Rosemont, IL, USA), and GAPDH (1:5,000, Proteintech, Rosemont, IL, USA) overnight at 4°C followed by horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Proteintech, Rosemont, IL, USA) at room temperature for 2 h. The blots were visualized with an enhanced chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology) and scanned by ChemImager 5500 v2.03 software.
Eif4a3
Manufactured by Proteintech
Sourced in United States
EIF4A3 is a protein that functions as an ATP-dependent RNA helicase. It is involved in the splicing and export of mRNA from the nucleus to the cytoplasm. EIF4A3 is a core component of the exon junction complex, which is crucial for various post-transcriptional regulatory processes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Ecl kit, by Beyotime (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Lamc2, by Abcam (1 mentions)
Ube3a, by Abcepta (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bx51 light microscope, by Olympus (1 mentions)
Caspase 3, by Abcam (1 mentions)
Aldh1a3, by Abcam (1 mentions)
Histone h3k9ac, by Cell Signaling Technology (1 mentions)
Ha tag, by Proteintech (1 mentions)
5 protocols using eif4a3
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis Workflow
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Cells were harvested and lysed by 1 × SDS buffer. Lysates were sonicated and centrifuged (13,000 rpm, 4 °C) for 10 min. Proteins were separated by 8–12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA). Membranes were immunoblotted with anti-rabbit NOP58 (1:1000), EIF4A3 (1:1000) (Proteintech, Chicago, USA; Abcam, UK) and anti-mouse LAMC2 (1:500), GAPDH (1:2000) (Abcam, UK; Zsbio, Beijing, China), and then were incubated with hybrid secondary antibody, and the data was collected by FluorChem V2.0 (Alpha Innotech Corp, USA).
3
Antibody Validation Protocols
4
Immunohistochemical Analysis of Tumor Samples
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Tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were then processed for H&E and IHC. Primary antibodies specific for Ki-67 (1: 1000, Abcam, USA), caspase 3 (1:100, Abcam, USA), EIF4A3 (1:200, Proteintech, China), OLFML3 (1:500, ThermoFisher Scientific, USA) and ALDH1A3(1: 200, Abcam, USA) were used. All images were recorded with an Olympus BX-51 light microscope.
5
Antibody Validation and Small Molecule Inhibitors
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The primary antibodies against METTL3 (#86132), AGO2 (#82897), Histone H3K9ac (#9649), Histone H4K5ac (#8647) were purchased from Cell Signaling Technology (Beverly, MA, USA), and antibodies against RNF113A (#27018-1-AP), EIF4A3 (#17504-1-AP), MDM2 (#66511-1-lg), P21 (#10355-1-AP), P53 (#10442-1-AP), HA-Tag (#66006-2-Ig), Flag (#66008-4-lg, #20543-1-AP) were obtained from proteintech (USA). Antibody against GAPDH (ab8245) was obtained from Abcam (Cambridge, UK). HRP-conjugated secondary antibodies, goat-anti-mouse-HRP (AB_2338504) and goat anti-rabbit-HRP (AB_2337938), were bought from Jackson ImmunoResearch. The METTL3 inhibitor STM2457, Chidamide, 5-Azacytidine, Ara-C, and MG132 were purchased from MCE (USA).
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