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. 2023 Mar 23;299(5):104644. doi: 10.1016/j.jbc.2023.104644

Figure 3.

Figure 3

Expression of GST-RagA and GST-RagC and isolation of mouse mTORC1. Coomassie stain (A) and SDS-page Western immunoblotting (B) of 8% acrylamide gel of recombinant human GST-RagA and GST-RagC expressed in Escherichia coli and purified on glutathione beads, as indicated. C, Coomassie stain (upper panel) and immunoblotting (lower panel) of Rag-isolated mTORC1 from MEF lysates. D–F, Rag-isolated mTORC1 from MEFs (D and E) or HEK293A (E and F) were immunoblotted against the indicated proteins. Experiments were repeated at least three times. In C–F, mTORC1 was isolated by Rag GST pull-down, lysed, and boiled. Lysates were prepared from six confluent 15-cm plates (C, D, and F) and six confluent 15-cm plates (∼14 mg of protein) (E). MEFs, mouse embryonic fibroblasts; mTORC1, mechanistic target of rapamycin complex 1.