Protein Extraction and Western Blotting

Cells were homogenized, and extracts were prepared using lysis buffer (NER buffer from the NER-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), 1× protease inhibitor cocktail, 1 μM microcystin-LR). The soluble fraction was recovered by centrifugation at 16,000 × g for 10 min at 4 °C. Protein concentration was measured with the BCA Protein Assay Kit (Pierce Biotechnology), and 30 μg of protein from each sample was resolved by SDS-PAGE. The resolved bands were transferred onto a nitrocellulose membrane and subjected to western blotting with the appropriate antibodies.
The following primary antibodies were used: mouse anti-CK1δ (C-8) (1/1000, Santa Cruz Biotechnology, sc-55553), goat anti-CK1δ (N-19) (1/500, sc-6475, Santa Cruz Biotechnology), rabbit-anti C-terminal Brd4 (1/2000)^{19 (link)}, rabbit anti-Brd4 (1/1000, A301-985A50, Bethyl Laboratories Inc.), rabbit anti-phospho-Brd4 492/494 (1/2000, ABE1453, Millipore), mouse anti-Gapdh (1/5000, NB300-221, Novus Biolechne), goat anti-CK2α (1/500, sc-6479, Santa Cruz Biotechnology), mouse anti-Flag (1/5000, A8592, Sigma), and rabbit anti-cyclin D1 (1/1000, ab134175, Abcam). The following secondary antibodies were used: anti-goat IgG–HRP (1/1000, 7074, Cell Signaling), anti-mouse IgG–HRP (1/1000, NXA931, GE Healthcare) and anti-rabbit IgG–HRP (1/1000, NA9340V, GE Healthcare).

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Penas C., Maloof M.E., Stathias V., Long J., Tan S.K., Mier J., Fang Y., Valdes C., Rodriguez-Blanco J., Chiang C.M., Robbins D.J., Liebl D.J., Lee J.K., Hatten M.E., Clarke J, & Ayad N.G. (2019). Time series modeling of cell cycle exit identifies Brd4 dependent regulation of cerebellar neurogenesis. Nature Communications, 10, 3028.

Publication 2019

NER-PER nuclear and cytoplasmic extraction reagents BCA Protein Assay Kit A8592 ab134175 NXA931 NA9340V

A301 Anti igg Antibodies Assay Brd4 Buffer Cells Centrifugation Ck2α Cyclin d1 Cytoplasmic Gapdh Goat Igg hrp Membrane Microcystin lr Mouse Nitrocellulose Novus Protease inhibitor Protein Rabbit Sds page

Corresponding Organization :

Other organizations : Sylvester Comprehensive Cancer Center, University of Miami, Rockefeller University, Florida International University, The University of Texas Southwestern Medical Center, University of Nebraska–Lincoln

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Variable analysis

independent variables

Lysis buffer (NER buffer from the NER-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific), 1× protease inhibitor cocktail, 1 μM microcystin-LR)

dependent variables

Protein expression of CK1δ, Brd4, phospho-Brd4, Gapdh, CK2α, Flag, and cyclin D1 measured by western blotting

control variables

Protein concentration measured with the BCA Protein Assay Kit (Pierce Biotechnology)
30 μg of protein from each sample resolved by SDS-PAGE

controls

Positive controls: Not explicitly mentioned.
Negative controls: Not explicitly mentioned.

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