Western Blot Analysis of Cell Signaling Proteins

Whole cell lysates (50 μg) were separated by SDS-PAGE and the proteins transferred to a nitrocellulose membrane for Western blotting as previously described (20 (link)). Specific primary antibodies were prepared according to the manufacturer’s instructions; Cyclin D1 (Cat. No. sc-753, clone H295, Santa Cruz, Santa Cruz, CA, USA), phospho Rb S780 (Cat. No. 9307S, Cell Signaling, Beverly, MA, USA), LC3-A/B (Cat. No. 4108, Cell Signaling, Danvers, MA, USA), AMPK (Cat. No. 2532S, Cell Signaling, Beverly, MA, USA), phospho AMPK T172 (Cat. No. 2531S, Cell Signaling, Beverly, MA, USA), Bnip3 (Cat. No. 3485-1, Epitomics, Burlingame, CA, USA) and Vinculin (Cat. No. V9131, Sigma-Aldrich, St. Louis, USA), which was used as an internal protein loading control. The LKB1 phospho-serine 325 (pS325) antibody was previously described (6 (link)). Anti rabbit (Cat. No. sc-2004, Santa Cruz, Santa Cruz, CA, USA) or mouse (Cat. No. sc-2005, Santa Cruz, Santa Cruz, CA, USA) secondary HRP-conjugated antibodies were diluted at 1:3000.

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Casimiro M.C., Di Sante G., Di Rocco A., Loro E., Pupo C., Pestell T.G., Bisetto S., Velasco-Velázquez M.A., Jiao X., Li Z., Kusminski C.M., Seifert E.L., Wang C., Ly D., Zheng B., Shen C.H., Scherer P.E, & Pestell R.G. (2017). Cyclin D1 restrains oncogene-induced autophagy by regulating the AMPK-LKB1 signaling axis. Cancer research, 77(13), 3391-3405.

Publication 2017

LC3-A/B phospho AMPK T172 Bnip3

Antibodies Antibody Cell Clone Cyclin d1 Lkb1 Membrane Mouse Nitrocellulose Protein Rabbit Sds page Serine Vinculin

Corresponding Organization : Baruch S. Blumberg Institute

Other organizations : Thomas Jefferson University, The University of Texas Southwestern Medical Center, Massachusetts General Hospital, Harvard University

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Variable analysis

independent variables

Primary antibodies used for Western blotting

dependent variables

Protein expression levels of Cyclin D1, phospho Rb S780, LC3-A/B, AMPK, phospho AMPK T172, Bnip3, and Vinculin

control variables

Total protein amount (50 μg) loaded per lane for Western blotting
Vinculin as an internal protein loading control

controls

Positive controls: None explicitly mentioned
Negative controls: None explicitly mentioned

Annotations

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