Protocol detail

Immunoblotting Assay for Protein Complexes

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Cell lysates were prepared with high salt lysis buffer (150 mM Tris-HCl [pH 7.9], 500 mM NaCl, 1% (w/v) Nonidet P-40). Immunoblotting was performed according to a standard procedure using the following primary antibodies: anti-GST (sc-138, Santa Cruz Biotechnology), anti-β-actin (ab6276, Abcam), anti-Aiolos (sc-101982, Santa Cruz Biotechnology), anti-GSPT1 (ab49878, Abcam), anti-DDB1 (ab97522, Abcam), anti-CUL4A (ab72548, Abcam), anti-ROC1 (ab86862, Abcam), and anti-CRBN (CRBN65 mAb, Celgene)^{34 (link)}.

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Tateno S., Iida M., Fujii S., Suwa T., Katayama M., Tokuyama H., Yamamoto J., Ito T., Sakamoto S., Handa H, & Yamaguchi Y. (2020). Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide. Scientific Reports, 10, 4012.

Publication 2020

ab6276, anti-Aiolos (sc-101982, ab49878 ab97522 ab72548

Actin Antibodies Buffer Cell Crbn Ddb1 Nacl Nonidet p 40 Tris

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Variable analysis

independent variables

High salt lysis buffer (150 mM Tris-HCl [pH 7.9], 500 mM NaCl, 1% (w/v) Nonidet P-40)

dependent variables

Protein expression levels of GST, β-actin, Aiolos, GSPT1, DDB1, CUL4A, ROC1, and CRBN

control variables

Standard immunoblotting procedure

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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