Western Blot Analysis of Cellular Fractions

Cells were collected and lysed in RIPA buffer, and analyzed by western blot as we described (13 (link), 75 (link)). Briefly, protein concentration was determined with Pierce BCA assay, and Western blot was conducted using Immobilon Western Chemiluminescent HRP (EMD Millipore) and quantified using the Thermo Fisher iBright Imaging system. Nuclear and cytoplasmic fractionation was performed using HAEC lysates with NE-PER^® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific 78833) per the manufacturer’s instruction. Antibodies used are listed in Table 3.

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Demos C., Johnson J., Andueza A., Park C., Kim Y., Villa-Roel N., Kang D.W., Kumar S, & Jo H. (2022). Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells. Frontiers in Cardiovascular Medicine, 9, 979745.

Publication 2022

Immobilon Western Chemiluminescent HRP iBright Imaging system NE-PER® Nuclear and Cytoplasmic Extraction Reagents

Antibodies Assay Buffer Cells Cytoplasmic Fractionation Immobilon Protein Ripa Western blot

Corresponding Organization : Emory University

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Variable analysis

independent variables

Nuclear and cytoplasmic fractionation

dependent variables

Protein concentration
Western blot signal quantification

control variables

RIPA buffer for cell lysis
Pierce BCA assay for protein concentration determination
Immobilon Western Chemiluminescent HRP for Western blot
Thermo Fisher iBright Imaging system for Western blot quantification
NE-PER® Nuclear and Cytoplasmic Extraction Reagents for nuclear and cytoplasmic fractionation

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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