Western Blot Analysis of Cell Signaling

For WB, cells were collected and lysed in RIPA buffer (Cell Signaling) with a complete protease inhibitor cocktail (Roche), phosphatase inhibitors (Roche), and PMSF (Sigma), then centrifuged at 12,000g for 10 min at 4 °C. Protein concentration was measured by Bio-Rad protein assay kit (Hercules, CA) according to the manufacturer’s instructions. Western blot assays were performed as previously described^{12 (link)}. The primary antibodies were obtained as follows: anti-caspase-3, anti-PCNA, anti-β-Catenin, anti- Non-phospho (Active) β-Catenin, and Exosomal Marker Antibody Sampler Kit antibody (Flotillin-1 and EpCAM) from Cell Signaling Technology, anti-β-actin antibody from Abmart.

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Xu H., Hao Z., Wang Y., Zhang D., Li J., Chen L., Yao N., Qian B., Peng X, & Zhan X. (2023). Liquid tumor microenvironment enhances WNT signaling pathway of peritoneal metastasis of gastric cancer. Scientific Reports, 13, 11125.

Publication 2023

RIPA buffer complete protease inhibitor cocktail phosphatase inhibitors PMSF protein assay kit anti-caspase-3 anti-PCNA anti-β-Catenin anti- Non-phospho (Active) β-Catenin anti-β-actin antibody

Actin Anti antibody Antibodies Antibody Assay Buffer Caspase 3 Cells Epcam Flotillin 1 Inhibitors Pcna Phosphatase Protease inhibitor Protein Rad protein Ripa Western blot assays β catenin

Corresponding Organization :

Other organizations : Second Military Medical University, Changhai Hospital, Shanghai CASB Biotechnology (China), Nantong University, Affiliated Hospital of Nantong University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Levels of caspase-3, PCNA, β-Catenin, Non-phospho (Active) β-Catenin, Flotillin-1, and EpCAM proteins

control variables

RIPA buffer (Cell Signaling) with a complete protease inhibitor cocktail (Roche), phosphatase inhibitors (Roche), and PMSF (Sigma)
Centrifugation at 12,000g for 10 min at 4 °C
Protein concentration measurement using Bio-Rad protein assay kit
β-actin as a loading control

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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