Western blotting was done as outlined in previous reports [1 ]. The primary antibodies included mouse anti-NEDD9 mAb (2G9) [1 ], -integrin β1, - pAP2 µ2 (Cell Signaling Technology), -integrin α1 (Abcam), -pTyr14/caveolin1, -caveolin1 (Santa Cruz Biotechnology), -GAPDH (ThermoFisher Scientific), -α-tubulin (Sigma-Aldrich), -Ras (EMD Millipore), -EGFR (eBioscience), - Transferin receptor, -adaptin-α, and -adaptin-γ (BD Bioscience). The secondary HRP-conjugated antibodies (Jackson Immuno Research Labs) were followed by using a chemiluminescent HRP Detection Reagent (Denville Scientific). The bands were digitized and quantified using a digital documentation and image analysis software (Syngene). For immunoprecipitation, the 2×106 cells were lysed in PTY buffer and incubated with 1µg of anti-NEDD9 mAb (2G9) or the control IgG overnight at +4°C as previously described [27 (link)].
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NEDD9 Immunoprecipitation and Western Blot
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Corresponding Organization : West Virginia University
Other organizations : Institute of Cytology
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- Primary antibodies: mouse anti-NEDD9 mAb (2G9), -integrin β1, - pAP2 μ2 (Cell Signaling Technology), -integrin α1 (Abcam), -pTyr14/caveolin1, -caveolin1 (Santa Cruz Biotechnology), -GAPDH (ThermoFisher Scientific), -α-tubulin (Sigma-Aldrich), -Ras (EMD Millipore), -EGFR (eBioscience), - Transferin receptor, -adaptin-α, and -adaptin-γ (BD Bioscience)
- Anti-NEDD9 mAb (2G9) or the control IgG used for immunoprecipitation
- Protein levels and phosphorylation status as measured by Western blotting
- Immunoprecipitation of NEDD9 protein
- Western blotting protocol as outlined in previous reports [1 (no link found)]
- Incubation of cell lysates with anti-NEDD9 mAb (2G9) or the control IgG overnight at +4°C for immunoprecipitation
- Positive control: None specified
- Negative control: Control IgG used for immunoprecipitation
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