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|Cells were collected and lysed in radioimmune precipitation assay buffer containing protease and phosphatase inhibitor mixtures at 4 °C for 30 min. Equal amounts of cellular proteins were analyzed by SDS-PAGE, followed by immunoblot. Anti-β-actin blot was used for the protein loading control. The heat shock procedure and GST pulldown were reported previously (29, 31). For starvation, NIH3T3 or U2OS cells were washed once with PBS and then incubated with EBSS for 30 min, 1 h, and 2 h. For rapamycin stimulation, cells were incubated with rapamycin or DMSO for 6 h or overnight.|
|Incubate cells at 30 min at 37 °C after adding the dye|
|Incase of Low dye staining in all treatments, increase the reagent concentration (500X dilution of the dye is recommended) and the incubation time.
Incase of High staining, it could be that the cell culture medium was depleted of nutrients and so change media 4-8 hours before the experiment.