Protein lysates (20–30 μg) were run onto 10% to 12% SDS–PAGE gels; electrophoresis and blocking were conducted as described (5 (link)). Blots were probed overnight at 4°C with respective antibodies. Primary antibodies anti-p-AKT (S473), anti-AKT, anti-Bcl-XL, anti-p-ERK (T202/Y204), anti-ERK, anti-p-STAT3, anti-STAT3, antisurvivin, and anti-β-actin were obtained from Cell Signaling Technology. Anti-EphB4 (clone# m265) and anti- ephrin-B2 antibodies were provided by Vasgene Therapeutics Inc.. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Sigma.
Protocol detail
Western Blot Analysis of Protein Signaling
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anti-Bcl-XL
anti-p-ERK (T202/Y204),
anti-ERK
anti-p-STAT3
anti-STAT3
antisurvivin
anti-β-actin
Horseradish peroxidase (HRP)-conjugated secondary antibodies
Actin
Anti antibodies
Antibodies
Clone
Electrophoresis
Ephrin b2
Gels
Horseradish peroxidase
Protein
Sds page
Stat3
Therapeutics
Corresponding Organization : University of Colorado Denver
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Protocol cited in 2 other protocols
Variable analysis
independent variables
- Protein lysates (20–30 μg)
- P-AKT (S473)
- Bcl-XL
- P-ERK (T202/Y204)
- P-STAT3
- STAT3
- Survivin
- EphB4 (clone# m265)
- Ephrin-B2
- β-actin
- Positive controls: Not explicitly mentioned.
- Negative controls: Not explicitly mentioned.
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