Western Blot Analysis of Autophagy Markers

Cells were lysed using 1% Triton in TBS containing protease and phosphatase inhibitors. Tumors were lysed using 1X RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of total protein were separated by electrophoresis on a 4–12% Bis-Tris gel and transferred to a PVDF membrane (23 (link)). Blots were blocked in 5% BSA, and then probed with antibodies against anti- NURR1 (sc-376984, SCB), anti- ATG 7 (10088-2AP, Proteintech), anti-ATG 12(D88H11, Cell Signaling Technology), anti-LC3BI/II(3868, Cell Signaling Technology), anti-Cleaved PARP (9532S, Cell Signaling Technology) and anti-α-tubulin (Sigma). Chemiluminescent (32106, Thermo Fisher Scientific) signal was captured using a Syngene G-BOX iChemi XT imager (26 (link)).

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Zarei M., Shrestha R., Johnson S., Yu Z., Karki K., Vaziri-Gohar A., Epps J., Du H., Suva L., Zarei M, & Safe S. (2021). Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma. Cancer Research Communications, 1(2), 65-78.

Publication 2021

anti-ATG 12(D88H11, anti-LC3BI/II(3868, anti-Cleaved PARP anti-α-tubulin Chemiluminescent G-BOX iChemi XT imager

Antibodies Bis tris Buffer Cells Electrophoresis Inhibitors Membrane Phosphatase Protease Protein Pvdf Ripa Tumors α tubulin

Corresponding Organization : Texas A&M University

Other organizations : Western University of Health Sciences, University School, Henan University of Science and Technology, Brigham and Women's Hospital, Harvard University

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Variable analysis

independent variables

Triton concentration (1%)
RIPA buffer (1X)

dependent variables

NURR1 protein levels
ATG7 protein levels
ATG12 protein levels
LC3BI/II protein levels
Cleaved PARP protein levels

control variables

Protease inhibitors
Phosphatase inhibitors
Total protein amount
Gel (4-12% Bis-Tris)
Membrane (PVDF)
Blocking (5% BSA)
Loading control (α-tubulin)

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