Propagation of 293T cells, transfection, preparation of cell extracts, and determination of protein concentration were performed as described previously44 (link),92 (link). The relative expression levels of A3B were determined by Western blot analysis of cell extracts (10 μg of total protein for each sample), using the Western Breeze chemiluminescent Western blot kit (Life Technologies, Grand Island, NY). The primary antibodies were anti-HA (Cell Signaling Technologies, Danvers, MA) for detection of all A3B proteins and anti-tubulin (Abcam, Cambridge, MA) for the loading control. The secondary antibodies, AlexaFluor 488 goat anti-rabbit IgG and AlexaFluor 680 goat anti-mouse IgG, were obtained from Life Technologies.
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Quantification of A3B Protein Expression
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Western Breeze chemiluminescent Western blot kit
anti-HA
anti-tubulin
AlexaFluor 488 goat anti-rabbit IgG
AlexaFluor 680 goat anti-mouse IgG
Anti igg
Antibodies
Cell extracts
Goat
Mouse
Propagation cells
Proteins
Rabbit
Transfection
Tubulin
Western blot
Western blot analysis
Corresponding Organization :
Other organizations : Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health
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Variable analysis
independent variables
- None explicitly mentioned
- Relative expression levels of A3B determined by Western blot analysis
- Propagation of 293T cells
- Transfection
- Preparation of cell extracts
- Determination of protein concentration
- Positive control: Anti-tubulin antibody as a loading control
- Negative control: Not explicitly mentioned
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