LC3B (D11) XP® Rabbit mAb

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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.

Protocol tips

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.

Publication protocol

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.

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