Analyzing Protein Expression via Immunoblotting

To analyze protein expression in cells, immunoblotting analysis was performed as previously described [2 (link)]. Antibodies against the following proteins were obtained from Santa Cruz Technology (California, US): Aur A (sc-25425), Aur B (sc-25426), BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), cyclin D1 (sc-718), cyclin E (sc-247), cyclin A (SC-751). Antibodies against BRCA2 (19791-1-AP) and cyclin B1 (cs-4135) were obtained from Proteintech (Chicago, USA) and Cell Signaling Technology (Massachusetts, US), respectively. The detection of β-actin (A2228, Sigma Aldrich, St. Louis, MO) was used as a loading control. The secondary antibodies were F(ab)2 fragments of donkey anti-mouse immunoglobulin or of donkey anti-rabbit immunoglobulin linked to horseradish peroxidase from Cell Signaling Technology (Massachusetts, US). Immunoblotting reagents were from an electrochemiluminescence kit (Amersham Biosciences). To test whether the expression levels of proteins were regulated through proteasome-mediated degradation, cells were exposed with 20 μM MG132 (#S2619, Selleck Company, Texas, America) for 3 h and then harvested for Western blotting analysis.

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Wang Y., Wang Z., Qi Z., Yin S., Zhang N., Liu Y., Liu M., Meng J., Zang R., Zhang Z, & Yang G. (2014). The negative interplay between Aurora A/B and BRCA1/2 controls cancer cell growth and tumorigenesis via distinct regulation of cell cycle progression, cytokinesis, and tetraploidy. Molecular Cancer, 13, 94.

Publication 2014

BRCA1 (sc-6954), CDK2 (sc-163), CDK4 (sc-260), CDK6 (sc-177), electrochemiluminescence kit

Actin Anti immunoglobulin Antibodies Brca1 Brca2 Cdk2 Cdk6 Cells Cyclin a Cyclin b1 Cyclin d1 Cyclin e Donkey Horseradish peroxidase Immunoglobulin fragments Mg132 Mouse Proteasome Proteins Rabbit Western blotting analysis

Corresponding Organization : Fudan University Shanghai Cancer Center

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Variable analysis

independent variables

Exposure of cells to 20 μM MG132 for 3 hours

dependent variables

Expression levels of the following proteins: Aur A, Aur B, BRCA1, BRCA2, CDK2, CDK4, CDK6, cyclin A, cyclin B1, cyclin D1, cyclin E

control variables

Detection of β-actin as a loading control

controls

Positive control: Not specified
Negative control: Not specified

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