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. 2023 May 23;6(8):e202201525. doi: 10.26508/lsa.202201525

Figure 8. RagA-C can rescue amino acid-dependent mTORC1 activation in MAP4K3 k.o. cells.

Figure 8.

(A) We transfected MAP4K3 HEK293A k.o. cells (M4 line) with RagA-C or pcDNA empty vector and maintained cells under baseline complete media (CM) conditions or subjected cells to amino acid starvation (−) or amino acid starvation followed by amino acid restimulation (+). We immunoblotted the resulting cell protein lysates for MAP4K3, RagA, phosphorylated S6, total S6, phosphorylated 4E-BP1, and total 4E-BP1, as indicated. Note rescue of mTORC1 activation in MAP4K3 k.o. cells expressing RagA-C in comparison with MAP4K3 k.o. cells expressing pcDNA empty vector (boxed). α-tubulin served as the loading control. (A, B) TOP: we quantified phosphorylated S6 and total S6 in (A) by densitometry, determined the ratio of phosphorylated S6: total S6, and normalized the results to pcDNA-transfected MAP4K3 k.o. cells in complete media. *P < 0.05, t test; n = 3 biological replicates. (A) BOTTOM: We quantified phosphorylated 4E-BP1 and total 4E-BP1 in (A) by densitometry, determined the ratio of phosphorylated 4E-BP1: total 4E-BP1, and normalized the results to pcDNA-transfected MAP4K3 k.o. cells in complete media. **P < 0.01, t test; n = 3 biological replicates. Error bars = s.e.m.