Extended Data Fig. 6: Asparagine Limitation Increases RTK Expression.
From: Translational reprogramming marks adaptation to asparagine restriction in cancer
a, Immunoblotting of EGFR in melanoma cells 72 hr after treatment with indicated si-ASNS. b, qRT-PCR analysis of EGFR transcript in melanoma cells 48 hr after treatment with si-ASNS. c-e, Immunoblotting of EGFR in melanoma cells 72 hr after treatment either individually or as a combination with si-ASNS and Torin 1 (c), si-ASNS and si-eIF4E (d), or si-ASNS and si-MNK1 (e). f, Immunoblotting of phospho and total ERK1/2 in melanoma cells treated 72 hr with si-ASNS, EGFR inhibitor (Gefitinib), or both. g, qRT-PCR analysis of VEGFR-2 transcript levels in melanoma cells 48 hr after treatment with si-ASNS#2 or #3. h, (left) Immunoblotting of VEGFR-2, PDGFR-B, α-Tubulin, and GAPDH in melanoma cells treated with NT-siRNA, si-ASNS#1 or #2 followed by L-azidohomoalanine (AHA) labeling (see Methods). (right) Ponseau-S staining of streptavidin pull-down fraction from melanoma cells left untreated or treated with AHA. i, Immunoblotting of VEGFR-2 and PDGFR-B in melanoma cells 72 hr after treatment with si-ASNS, ISRIB, or both. j, Immunoblotting of the indicated proteins in melanoma cells 72 hr after treatment with si-ASNS, si-GCN2, or both. Data are representative of three independent experiments and presented as the mean ±SEM of n=3 technical replicates in b and g.
