Total cellular protein was extracted using RIPA buffer (Sigma-Aldrich) and quantified with BCA assay (Sigma-Aldrich). SPC25 and PDGF were measured with ELISA kits from MyBiosource and R&D Biosystem, respectively. Western blot was performed with the following antibodies: a rabbit anti-human SPC25 (1:750; Ab236972, Abcam, Dallas, TX, USA), a mouse anti-human Egr-1 (1:2,000; Ab55160, Abcam), a rabbit anti-human PDGF (1:1,000; Ab23914, Abcam), and a mouse anti-human GAPDH antibody (1:1,000; Ab8245, Abcam). All secondary antibodies were from Jackson ImmunoResearch Labs (West Grove, PA, USA). Quantification was done with ImageJ (NIH, Bethesda, MA, USA). Immunocytochemistry was done with a mouse anti-human PDGFR alpha antibody (1:50; Ab96569, Abcam).
Ab55160
Manufactured by Abcam
Sourced in United States
Ab55160 is a lab equipment product offered by Abcam. It is a device designed for laboratory use, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Additional information about the intended use or capabilities of this product is not available.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab23914, by Abcam (1 mentions)
Ab96569, by Abcam (1 mentions)
Bca assay, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab8245, by Abcam (1 mentions)
Elisa kit, by MyBioSource (1 mentions)
Ab118929, by Abcam (1 mentions)
Ab53036, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Phospho ser133creb, by Cell Signaling Technology (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Nuclear complex co ip kit, by Active Motif (1 mentions)
5 protocols using ab55160
1
Protein Quantification and Analysis
2
ChIP-PCR Assay for EGR1-PFKFB3 Interaction
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The ChIP-PCR procedure was similar to previously described procedures [30 (link)]. ChIP DNA from anti-EGR1 (ab55160, Abcam)-treated cells was used to examine the association between EGR1 and PFKFB3. DNA from anti-IgG antibody-treated cells served as the control. Purified DNA was used to analyze the PFKFB3 proximal promoter region by real-time PCR. The PFKFB3 primers were as follows: forward: 5′-TGTGAAAACCAGATGCCAGC-3′; and reverse: 5′-GGACTTGAACTGAGCCTTGC-3′. The relative amplification of the promoter sequence of the gene was calculated using the 2−ΔΔCT method.
3
Westerns for Hippocampal Protein Analysis
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Protein was extracted from the hippocampi in rats with etomidate treatment or vehicle according to a previously described procedure.[14 (link)] Membranes were probed with primary antibodies against CREB, phospho-Ser133-CREB (#9104 and #9191, respectively, Cell Signaling, USA), Arc, c-fos, and Egr1 (ab118929, ab53036, and ab55160, respectively, Abcam, USA). Horseradish peroxidase conjugated anti-rabbit or anti-mouse secondary antibodies were used and visualized using the enhanced chemiluminescence kit (Thermo Scientific, USA). The density of each band was quantified using the ImageJ software (National Institutes of Health, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab1603, Abcam) was used as a loading control.
4
Western Blot Analysis of Key Proteins
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Tissues were lysed in RIPA buffer (Beyotime, Shanghai, China) containing 1 mmol/l PMSF, and the protein concentrations were determined with a BCA protein assay kit (Thermo, Rockford, IL, USA). Thirty micrograms of protein was separated by 10% SDS–PAGE (Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to NC membranes. After being incubated with blocking buffer (5% nonfat dry milk in TBST), the membranes were incubated overnight at 4 °C with the following primary antibodies: PFKFB3 (13763-1-AP, Proteintech), IGFBP5 (55205-1-AP, Proteintech), EGR1 (ab55160, Abcam), β-actin (ab8226, Abcam), and tubulin (66240-1, Proteintech).
5
EGR1 Interaction with HTLV-1 Tax
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293T cells were co-transfected with pCMV-Tax, pCMV-M22Tax, or pCMV-M47 Tax and/or pcDNA3.0-EGR1 for 48 h. Cells were harvested and interactions between EGR1 and Tax were detected using immune co-precipitation (Nuclear Complex Co-IP Kit; 54001, Active Motif). An EGR1 monoclonal antibody (ab55160, Abcam) was used to capture EGR1 protein and the target protein, Tax, was detected via western blot.
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