P lkb1 s 428

Manufactured by Cell Signaling Technology

P-LKB1 (S-428) is an antibody that recognizes the phosphorylated form of LKB1 at serine 428. LKB1 is a serine/threonine kinase that plays a crucial role in the regulation of cellular energy metabolism and polarity.

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P-LKB1 (16 citations)

3 protocols using p lkb1 s 428

Immunoblotting Analysis of Signaling Proteins

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Cell lysis and Immunoblotting was performed as described previously (8 (link)). 30 μg of protein was loaded per lane in 8–10% SDS gels. The following antibodies were used to probe the membranes: p-ACC (S-79) (#11818 Cell signaling), p-mTOR (S-2448) (#ab109268 Abcam), mTOR (#ab2732 Abcam), p-LKB1 (S-428) (#3482 Cell signaling), LKB1 (#3050 Cell signaling), p-AMPK (T-172) (#2535 Cell signaling), p-AMPK (S-173) (#ab55886 Abcam), GAPDH (#2118 Cell signaling), Vinculin (sc-5573, Santa Cruz Biotechnologies), p-TAK1 (#4508 Cell signaling), TAK1 (#5206 Cell signaling), p-CAMKII (#12716 Cell Signaling). For Figures 1, 5, Western blots were developed using near-IR-labeled secondary antibodies and quantitated on a Li-COR Odyssey CLx and analyzed using ImageStudio v5.2. Other blots were developed on film and analyzed semi-quantitatively. All in vitro experiments were repeated 2–4 times with consistent results observed.

Kari S., Vasko V.V., Priya S, & Kirschner L.S. (2019). PKA Activates AMPK Through LKB1 Signaling in Follicular Thyroid Cancer. Frontiers in Endocrinology, 10, 769.

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Analysis of Cellular Signaling Pathways

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Antibodies against LKB1, pLKB1^S428, AMPK, pAMPK^Thr172, p70S6K, pp70S6K^Thr389, S6 Ribo, pS6 Ribo^S235/236, pS6 Ribo^S240/244, ULK1, pULK1^S555, pULK^S757, LC-3B, cyclin A, cyclin D1, and PARP were purchased from Cell Signaling. Antibodies for p27, cyclin B1, and CDC25C were from Santa Cruz Biotechnology, and β-actin from Sigma-Aldrich. Secondary antibodies were purchased from GE Healthcare. Cells were treated with indicated concentrations of hMGL-4.0 for the specified time. After incubation, cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer. Proteins were separated by using 4 to 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. After blocking in 5% BSA for 1 h, the membranes were probed with specific primary antibodies (listed above) overnight at 4 °C. Following secondary antibody incubation, the membranes were visualized using a commercially available chemiluminescent detection kit (Pierce Biotechnology).

Lu W.C., Saha A., Yan W., Garrison K., Lamb C., Pandey R., Irani S., Lodi A., Lu X., Tiziani S., Zhang Y.J., Georgiou G., DiGiovanni J, & Stone E. (2020). Enzyme-mediated depletion of serum l-Met abrogates prostate cancer growth via multiple mechanisms without evidence of systemic toxicity. Proceedings of the National Academy of Sciences of the United States of America, 117(23), 13000-13011.

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Western Blot Analysis of Signaling Proteins

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Cells were washed with ice-cold PBS and lysed with RIPA lysis buffer. Proteins in the supernatants were separated by 4–20% SDS-polyacrylamide gel electrophoresis and transferred to 0.45 μm nitrocellulose membranes. After blocking, the membranes were incubated with the appropriate primary antibodies against IFT88 (ProSci, Poway, CA), LKB1, p-LKB1(S428), AKT, p-AKT(S473), PTEN and p-PTEN(Ser380/Thr382/Thr383) (Cell Signaling), FAK (Cell Signaling) and GAPDH (ProSci) at 4°C overnight. After washing, the blots were subsequently incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies. Protein signals were developed using the Clarity Western ECL Substrate Bio-Rad (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were repeated at least three times.

Mansini A.P., Peixoto E., Jin S., Richard S, & Gradilone S.A. (2019). The chemosensory function of primary cilia regulates cholangiocyte migration, invasion and tumor growth. Hepatology (Baltimore, Md.), 69(4), 1582-1598.

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