Quantitative Western Blot Analysis of Apoptosis and Wnt Signaling

Western blot of whole protein lysates or nuclear proteins from frozen tumor tissues or cells was performed as previously described (24 (link)). Antibodies for cleaved caspase 3 (Asp175) (5A1E), cleaved PARP (Asp214) (D64E10), cyclin D1 (92G2), β-catenin (6B3), active β-catenin (D13A1), TCF4 (C48H11), axin 1 (C95H11), tubulin (DM1A) and GAPDH (D16H11) were purchased from Cell Signaling Technology. Other antibodies included lamin B1 (ab16048, Abcam), COP1 (ab56400, Abcam), tankyrase1/2 (H-350, Santa Cruz), and active β-catenin (Clone 8E7, Millipore). ImageJ software was used to measure the relative density for signaling expression.

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Zeng S., Seifert A.M., Zhang J.Q., Cavnar M.J., Kim T.S., Balachandran V.P., Santamaria-Barria J.A., Cohen N.A., Beckman M.J., Medina B., Rossi F., Crawley M.H., Loo J.K., Maltbaek J.H., Besmer P., Antonescu C.R, & DeMatteo R.P. (2017). Wnt/β-catenin signaling contributes to tumor malignancy and is targetable in gastrointestinal stromal tumor. Molecular cancer therapeutics, 16(9), 1954-1966.

Publication 2017

GAPDH (D16H11) ab16048 ab56400, active β-catenin (Clone 8E7,

Antibodies Caspase 3 Cells Clone Cop1 Cyclin d1 Frozen Gapdh Lamin b1 Nuclear proteins Protein Tcf4 Tissues Tubulin Tumor Western blot β catenin

Corresponding Organization : Memorial Sloan Kettering Cancer Center

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Variable analysis

independent variables

Western blot of whole protein lysates or nuclear proteins from frozen tumor tissues or cells

dependent variables

Relative density for signaling expression of cleaved caspase 3 (Asp175), cleaved PARP (Asp214), cyclin D1 (92G2), β-catenin (6B3), active β-catenin (D13A1), TCF4 (C48H11), axin 1 (C95H11), tubulin (DM1A) and GAPDH (D16H11)

control variables

Antibodies for cleaved caspase 3 (Asp175), cleaved PARP (Asp214), cyclin D1 (92G2), β-catenin (6B3), active β-catenin (D13A1), TCF4 (C48H11), axin 1 (C95H11), tubulin (DM1A) and GAPDH (D16H11) from Cell Signaling Technology
Antibodies for lamin B1 (ab16048, Abcam), COP1 (ab56400, Abcam), tankyrase1/2 (H-350, Santa Cruz), and active β-catenin (Clone 8E7, Millipore)

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