Protocol detail

Western Blot Analysis of Subcellular Proteins

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For Western blot analysis, cells were harvested and lysed in RIPA lysis buffer (Cell Signal Technology). To investigate subcellular distribution of proteins, nuclear and cytoplasmic fractions were enriched using Nuclear and Cytoplasmic Protein Extraction Kit (CoWin Bioscience). Western blot assays were followed according to an established protocol (20 (link)). Antibody used included mouse anti-GFP (Abmart), rabbit anti-acetyl Lysine (Abcam), rabbit anti-SUMO1 (Abcam), rabbit anti-Lamin B1 (Abcam), mouse anti-Ub (Santa Cruz), rabbit anti-USP49 (NOVUS), mouse anti-Flag (Proteintech), mouse anti-GAPDH (Thermo Fisher Scientific), mouse anti–β-tubulin (Thermo Fisher Scientific), rabbit anti-GRα (Abcam), rabbit anti-GRβ (Abcam). For protein stability detections, cells were treated with proteasome inhibitor MG132 (20 μmol/L, Sigma-Aldrich) for 6 hours or protein synthesis inhibitor Cycloheximide (CHX, 100 μg/mL, Sigma-Aldrich) for indicate time before harvest.

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Hu Y., Jiang Y., Zhang Z., Wang J., Zhang B., Gong L., Ji L., Pu Z., Yang X., Zou J, & Yin Y. (2021). Oncogenic Activity of Glucocorticoid Receptor β Is Controlled by Ubiquitination-Dependent Interaction with USP49 in Glioblastoma Cells. Molecular Cancer Research, 20(1), 92-101.

Publication 2021

RIPA lysis buffer mouse anti-GFP rabbit anti-acetyl Lysine rabbit anti-SUMO1 rabbit anti-Lamin B1 mouse anti-Ub mouse anti-Flag mouse anti-GAPDH mouse anti–β-tubulin MG132 Cycloheximide (CHX,

Anti b1 Antibody Buffer Cells Cycloheximide Cytoplasmic Gapdh Lamin Lysine Mg132 Mouse Novus Proteasome inhibitor Protein Protein synthesis inhibitor Rabbit Ripa Sumo1 Tubulin Western blot assays

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Variable analysis

independent variables

Treatment with proteasome inhibitor MG132 (20 μmol/L) for 6 hours
Treatment with protein synthesis inhibitor Cycloheximide (CHX, 100 μg/mL) for an indicated time

dependent variables

Subcellular distribution of proteins
Protein stability

control variables

RIPA lysis buffer (Cell Signal Technology) for cell lysis
Nuclear and Cytoplasmic Protein Extraction Kit (CoWin Bioscience) for subcellular fractionation
Established Western blot protocol (20)

positive controls

Mouse anti-GAPDH
Mouse anti–β-tubulin

negative controls

Not explicitly mentioned

Annotations

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