Protocol detail

Western Blotting of Vascular Smooth Muscle Cells

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Western blotting assay was conducted as previously described (17 (link)). Extractions of VSMCs and artery tissues were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai). Protein samples were segregated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a poly-vinylidene fluoride membrane (Millipore, Billerica, MA). Primary antibodies against VRK1, β-catenin, phospho (p)-mTOR^Ser2448, mTOR, p-S6^Ser235/236, S6, p-4EBP1^Thr37/46, 4EBP1, Caspase 3, Caspase 9 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA). Quantitative analysis of protein band was performed by using Image J software (Bethesda, MA).

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Sun X., Zhao W., Wang Q., Zhao J., Yang D, & Yang Y. (2022). Inhibition of VRK1 suppresses proliferation and migration of vascular smooth muscle cells and intima hyperplasia after injury via mTORC1/β-catenin axis. BMB Reports, 55(5), 244-249.

Publication 2022

RIPA buffer poly-vinylidene fluoride membrane β-catenin p-S6Ser235/236 p-4EBP1Thr37/46 4EBP1 Caspase 3 Caspase 9 GAPDH

4ebp1 Antibodies Artery Buffer Caspase 3 Caspase 9 Gapdh Membrane Mtor Poly vinylidene fluoride Protein Ripa Sodium dodecyl sulfate polyacrylamide gel electrophoresis Tissues Western blotting assay β catenin

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Variable analysis

independent variables

Extractions of VSMCs and artery tissues

dependent variables

Protein expression levels of VRK1, β-catenin, phospho (p)-mTOR^Ser2448^, mTOR, p-S6^Ser235/236^, S6, p-4EBP1^Thr37/46^, 4EBP1, Caspase 3, and Caspase 9

control variables

GAPDH (used as a loading control)

controls

No positive or negative controls were explicitly mentioned in the input protocol.

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