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License: CC BY
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Raw Western Blot Data for Protein X Expression in Condition Y

Authors: Jiang, Hai;

Raw Western Blot Data for Protein X Expression in Condition Y

Abstract

The primary antibodies used were as follows: FBXW11 (A7784, ABclonal), HIC1 (24949-1-AP, Proteintech), IRF1 (A7692, ABclonal), PARP (9542, CST), cleaved PARP (#5625, CST), caspase-3 (9662, CST), cleaved caspase-3 (9661, CST), IL-1β (12703, CST), cleaved IL-1β (83186, CST), Actin (AC004, ABclonal), and Anti-Ubiquitin (ab7780, Abcam). The membranes were washed three times with TBST, each for 10 minutes, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit IgG H&L, HRP, ab97051, 1:2000, Abcam, Cambridge, UK) for 1 hour. After TBST washes, the membranes were placed on a clean glass plate. Pierce™ ECL detection reagent (product number 32209, Thermo) was prepared by mixing equal volumes of solutions A and B in the dark, and the mixture was applied to the membranes. Imaging was performed using the Bio-Rad imaging system (ChemiDoc™ XRS+, BIO-RAD). For the WB experiment, total proteins were extracted from cells using RIPA lysis buffer containing PMSF (P0013B, Beyotime, Shanghai). Protein concentrations were determined with a BCA Protein Assay Kit (T23225, Thermo Fisher Scientific, Rockford, IL, USA). 50 μg of protein was dissolved in 2× SDS loading buffer, boiled at 100°C for 5 minutes, and then subjected to SDS-PAGE for electrophoresis. The proteins were wet-transferred onto a PVDF membrane, followed by blocking with 5% non-fat milk at room temperature for 1 hour. The membrane was then incubated overnight at 4°C with primary antibodies. The primary antibodies used included: FBXW11 (ABCLONAL, A7784), HIC1 (Proteintech, 24949-1-AP), IRF1 (ABCLONAL, A7692), PARP (CST, 9542), cleaved PARP (CST, 5625), caspase-3 (CST, 9662), cleaved caspase-3 (CST, 9661), IL-1β (CST, 12703), cleaved IL-1β (CST, 83186), Actin (ABCLONAL, AC004), and Anti-Ubiquitin (Abcam, ab7780). After primary antibody incubation, the membrane was washed thrice with TBST, each lasting 10 minutes. The membrane was then incubated with an HRP-conjugated secondary antibody (goat anti-rabbit IgG H&L, HRP, Abcam, ab97051, 1:2000) for 1 hour. For detection, the membrane was treated with the Pierce™ ECL detection reagent (product number 32209, Thermo) by mixing equal volumes of solutions A and B and applying the mixture onto the membrane. Imaging was performed using a Bio-Rad imaging system (ChemiDoc™ XRS+, BIO-RAD).

Keywords

Western blot

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selected citations
These citations are derived from selected sources.
This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Citations provided by BIP!
popularity
This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
BIP!Popularity provided by BIP!
influence
This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
BIP!Influence provided by BIP!
impulse
This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.
BIP!Impulse provided by BIP!
0
Average
Average
Average