Protein Expression Analysis by Western Blot

Western blotting to analyze protein expression was performed as previously described^{44 (link), 45 (link)}. Briefly, cell pellets and tissues were lysed using IP lysis buffer (Fisher Scientific) and the protein concentration was determined by a Bicinchoninic Acid Protein Assay (Thermo Scientific) using a FLUOstar Omega microplate reader (BMG Labtech). 25 μg of protein was used for immunoblot analysis using antibodies against DHODH (1:1,000, 14877-1-AP, Proteintech), GPX4 (1:1,000, MAB5457, R&D systems), vinculin (1:3,000, V4505, Sigma-Aldrich), SLC7A11 (1:1,000, 12691, Cell Signaling), ACSL4 (1:1,000, sc-271800, Santa Cruz), COQ2 (1:1,000, sc-517107, Santa Cruz Biotechnology), FSP1 (1:1,000, sc-377120, Santa Cruz Biotechnology), and Flag (1:10,000, 66008-3-Ig, Proteintech).

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Mao C., Liu X., Zhang Y., Lei G., Yan Y., Lee H., Koppula P., Wu S., Zhuang L., Fang B., Poyurovsky M.V., Olszewski K, & Gan B. (2021). DHODH-mediated ferroptosis defense is a targetable vulnerability in cancer. Nature, 593(7860), 586-590.

Publication 2021

IP lysis buffer Bicinchoninic Acid Protein Assay FLUOstar Omega microplate reader MAB5457 V4505 sc-271800 sc-517107 sc-377120

Antibodies Assay Bicinchoninic acid Buffer Cell Dhodh Gpx4 Immunoblot analysis Pellets Protein Protein a Tissues Vinculin

Corresponding Organization :

Other organizations : The University of Texas MD Anderson Cancer Center, The University of Texas Health Science Center at Houston

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Variable analysis

independent variables

Cell pellets and tissues were lysed using IP lysis buffer (Fisher Scientific)

dependent variables

Protein expression levels of DHODH, GPX4, vinculin, SLC7A11, ACSL4, COQ2, and FSP1

control variables

Protein concentration was determined by a Bicinchoninic Acid Protein Assay (Thermo Scientific) using a FLUOstar Omega microplate reader (BMG Labtech)

controls

Positive control: Not specified
Negative control: Not specified

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