RPPA analysis and antibody validation were performed as previously described (31 (link) ). Briefly, protein lysate was collected from subconfluent HNSCC cell cultures after 24 hours in full-serum medium (10% FBS). Protein lysates were adjusted to a 1-μg/μL concentration, and a serial dilution of 5 concentrations was printed, with 10% of the samples replicated for quality control (2470 Arrayer; Aushon Biosystems, Burlington, MA) on nitrocellulose-coated slides (Grace Bio-Labs, Bend, OR). Immunostaining was performed using a DakoCytomation-catalyzed system and diaminobenzidine colorimetric reaction. Spot intensities were analyzed and quantified using MicroVigene software (VigeneTech Inc., Carlisle, MA) to generate spot signal intensities.
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RPPA Analysis of HNSCC Cell Cultures
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Corresponding Organization : The University of Texas MD Anderson Cancer Center
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Variable analysis
independent variables
- Cell culture conditions (full-serum medium, 10% FBS)
- Protein lysate concentration (1-μg/μL)
- Spot signal intensities
- Subconfluent HNSCC cell cultures
- Protein lysate collection after 24 hours
- Serial dilution of 5 protein lysate concentrations
- 10% of samples replicated for quality control
- Nitrocellulose-coated slides
- Immunostaining using DakoCytomation-catalyzed system and diaminobenzidine colorimetric reaction
- Spot intensity analysis and quantification using MicroVigene software
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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