LMPTP inhibitor Compd. 23 was generated as described (14 (link)) and formulated in rodent chow at 0.1% (w/w) by Research Diets. CRISPR-Cas9 plasmids were purchased from ATUM. Pierce d -Luciferin monopotassium salt was purchased from Thermo Fisher Scientific and reconstituted using phosphate-buffered saline (PBS). For in vitro experiments, docetaxel and cabazitaxel were purchased from Selleckchem and reconstituted in sterile dimethyl sulfoxide (DMSO). The anti–phospho–eIF2-Ser51 (#3398), anti-eIF2 (#9722), anti-NRF2 (#12721), anti-ATF4 (#11815), anti–glyceraldehyde 3-phosphate dehydrogenase (#5174), anti–phospho-H2AX (#9718), anti-H2AX (#2595), and anti-pTyr (pY1000; #8954) antibodies were purchased from Cell Signaling Technology (CST). The rabbit anti-LMPTP antibody was described in (52 (link)). The mouse anti-LMPTP antibody (#sc-100343) was purchased from Santa Cruz Biotechnology. The anti-GSS antibodies were purchased from Thermo Fisher Scientific (#PA5-89891) or Santa Cruz Biotechnology (#sc-166882). Anti-rabbit (#NA934-1ML) and anti-mouse (#NA931-1ML) secondary antibodies were purchased from Thermo Fisher Scientific. TrueBlot anti-rabbit (#RL18-8816-31) and anti-mouse (#RL18-8817-31) secondary antibodies were purchased from Rockland. GSH-MEE (#353905) was purchased from Sigma-Aldrich. Unless otherwise specified, chemicals and other reagents were purchased from Sigma-Aldrich.
Protocol detail
Optimization and Validation of LMPTP Inhibitor
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Corresponding Organization : Cedars-Sinai Medical Center
Other organizations : The University of Texas at Austin, The University of Texas MD Anderson Cancer Center, Dartmouth College, Cotton (United States), Livestrong Foundation, Memorial Sloan Kettering Cancer Center, Lawrence Berkeley National Laboratory, Rice University, Caris Life Sciences (United States)
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Variable analysis
independent variables
- LMPTP inhibitor Compd. 23
- CRISPR-Cas9 plasmids
- Docetaxel
- Cabazitaxel
- Phospho-eIF2-Ser51 expression
- EIF2 expression
- NRF2 expression
- ATF4 expression
- Glyceraldehyde 3-phosphate dehydrogenase expression
- Phospho-H2AX expression
- H2AX expression
- PTyr (pY1000) expression
- LMPTP expression
- GSS expression
- Phosphate-buffered saline (PBS)
- Sterile dimethyl sulfoxide (DMSO)
- Pierce d-Luciferin monopotassium salt
- GSH-MEE
- None specified
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