Protein Isolation and Western Blot Analysis

Total protein (~20 μg) was isolated from cells following 72 h treatment or vehicle control (0.02% DMSO or ethanol) for protein analysis as previously described (10 (link), 14 (link)). The following antibodies were used: ASCT2 (#5345), SNAT1/SLC38A1 (#36057), EAAT2/SLC1A2 (#3838), LAT1/SLC7A5 (#5347), phospho-p70S6K (T389) (#9234), p70SK (#9202), phospho-mTOR(S2448) (#5536), phospho-mTOR(S2481) (#2974), mTOR (#2983), phospho-AKT(S473) (#4058), AKT (#4691), ATG13 (#13273) were from Cell Signaling, Danvers, MA; SNAT2/SLCA2 (#bs-12125R) was from Bioss, Woburn, MA; phospho-ATG13(S318) (#NBP2-19127) was from Novus, Centennial, CO; loading control antibodies such as actin (#sc-47778) was from Santa Cruz Biotechnology, Santa Cruz, CA and β-tubulin (#T7816) was from Sigma.

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Demas D.M., Demo S., Fallah Y., Clarke R., Nephew K.P., Althouse S., Sandusky G., He W, & Shajahan-Haq A.N. (2019). Glutamine Metabolism Drives Growth in Advanced Hormone Receptor Positive Breast Cancer. Frontiers in Oncology, 9, 686.

Publication 2019

ASCT2 LAT1/SLC7A5 phospho-p70S6K (T389) phospho-mTOR(S2448) phospho-mTOR(S2481) phospho-AKT(S473) ATG13 actin β-tubulin

Actin Antibodies Cells Dmso Ethanol Mtor Novus P70s6k Protein Slc1a2 Slc7a5 Tubulin

Corresponding Organization : Georgetown University

Other organizations : Calithera (United States), Indiana University Bloomington, Indiana University – Purdue University Indianapolis, Virginia Tech

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Variable analysis

independent variables

72 h treatment
Vehicle control (0.02% DMSO or ethanol)

dependent variables

Total protein (~20 μg) isolated from cells
Protein expression levels of ASCT2, SNAT1/SLC38A1, EAAT2/SLC1A2, LAT1/SLC7A5, phospho-p70S6K (T389), p70SK, phospho-mTOR(S2448), phospho-mTOR(S2481), mTOR, phospho-AKT(S473), AKT, ATG13, phospho-ATG13(S318)

control variables

Cells
Experimental methods for protein isolation and analysis as previously described (10 (link), 14 (link))

positive controls

Not explicitly mentioned

negative controls

Vehicle control (0.02% DMSO or ethanol)

Annotations

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