Whole cell lysates from lung tumors isolated from mice were prepared as previously described7 (link). Briefly, tumors were homogenized in buffer containing phosphatase and protease inhibitors (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM pyrophosphate, 1 mM sodium orthovanadate, protease inhibitor tablet), centrifuged and supernatant was normalized, aliquoted and stored in −80°C freezer. Lysates were separated on 4–12% Bis-Tris protein gels (Thermo), transferred to PVDF membrane and probed with the following antibodies: SP-C (1:5000, AB3786 Milipore); Glut1 (1:2000, GT11-A, Alpha Diagnostic); Ndufs1 (1:2000, ab169540, abcam); Ndufs1 (1:2000, sc-271510, Santa Cruz); Ndusv1 (1:500, 11283–1-AP, Proteintech), Ndufv2 (1:2000, sc-271620, Santa Cruz), Tom20 (1:10000, FL-145, Santa Cruz), Tom40 (1:2000, 18409–1-AP, Proteintech); Tom70 (1:2000, 14528–1-AP, Proteintech); Tim23 (1:2000, 11123–1-AP, Proteintech); actin (1:8000, #4970 and #3700, Cell Signaling Technology). Intensity of bands was quantified using Image J.
Protocol detail
Lung Tumor Lysate Preparation and Analysis
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Actin
Antibodies
Bis tris
Buffer
Cell
Diagnostic
Edta
Egta
Gels
Glut1
Lung tumors
Membrane
Mice
Nacl
Orthovanadate
Phosphatase
Protease inhibitor
Protein
Pvdf
Pyrophosphate
Sodium
Sodium fluoride
Tablet
Tom70
Tris
Triton x 100
Tumors
Corresponding Organization :
Other organizations : University of California, Los Angeles, University of North Carolina at Chapel Hill, Regis College, Loyola Marymount University
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Variable analysis
independent variables
- None explicitly mentioned
- Intensity of protein bands quantified using ImageJ
- Whole cell lysates from lung tumors isolated from mice
- Homogenized in buffer containing phosphatase and protease inhibitors (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 2.5 mM pyrophosphate, 1 mM sodium orthovanadate, protease inhibitor tablet)
- Centrifuged and supernatant normalized, aliquoted and stored in -80°C freezer
- Lysates separated on 4-12% Bis-Tris protein gels (Thermo) and transferred to PVDF membrane
- Probed with specific antibodies: SP-C, Glut1, Ndufs1 (2 antibodies), Ndusv1, Ndufv2, Tom20, Tom40, Tom70, Tim23, actin
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
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