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. 2021 May 13;184(10):2649–2664.e18. doi: 10.1016/j.cell.2021.03.031

Figure 3.

Figure 3

Cytoplasmic EML4-ALK protein granules locally activate RAS

(A–D) RAS-GTP levels of KRAS wild-type (A and C) or C185S cytosolic mutant (B and D) stably expressed in 293T or H3122 cells, followed by transfection of empty vector (EV), EML4-ALK, or oncogenic EGFR (A and B) or ±2 h of 100 nM crizotinib (C and D). n = 3. See STAR Methods for normalization details.

(E) Live-cell imaging of RAS-GTP reporter in Beas2B cells ± mTagBFP2::EML4-ALK. Arrow indicates a representative EML4-ALK granule with local enrichment of RAS-GTP (multiple non-highlighted granules also show colocalization).

(F) Per cell quantification of EML4-ALK granule/RAS-GTP reporter colocalization. WT denotes unmodified GFP-RBD reporter, RBD mutant (R59A/N64D) displays diminished RAS-GTP binding. n = 3, 30+ cells per replicate.

For all panels, error bars represent ± SEM, ∗p < 0.05, ∗∗p < 0.01, n.s., non-significant comparison, one-way ANOVA with post hoc Tukey’s HSD test (A, B, and F) or paired t test (C and D).

See also Figure S3.