Degradation Experiments Sample Clauses

Degradation Experiments. HEK-293T/17 cells were plated and transfected as described above with 2µg of receptor and 1 µg of empty vector, HA-ubiquitin or HA-ß-arrestin2. For ubiquitination experiments, after 24 h post-transfection, cells were treated overnight with 100nM MG-132 (Tocris) to inhibit the proteasome. Cells were washed, harvested, and solubilized as described above 48 h post-transfection. Cleared lysates were incubated with anti-HA agarose beads (Sigma) for 1h, washed, and eluted in Laemmli buffer. Tagged constructs were detected via Western blotting with mouse HRP-conjugated anti-Flag (Sigma) rabbit HRP- conjugated anti-HA (Abcam).
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Related to Degradation Experiments

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