Figure 7.

Signaling architecture of membraneless RTK protein granules

(A) List of EML4-ALK granule components.

(B) Anti-FLAG co-IP of FLAG-tagged EML4-ALK WT or ΔTD mutant expressed in 293T cells, followed by western blotting.

(C) Live-cell imaging of mTagBFP2::EML4-ALK and mEGFP-tagged signaling proteins expressed in Beas2B cells.

(D) List of CCDC6-RET granule components.

(E) Anti-FLAG co-IP of FLAG-tagged CCDC6-RET WT or ΔCC mutant expressed in 293T cells, followed by western blotting.

(F) Live-cell imaging of mTagBFP2::CCDC6-RET and mEGFP-tagged signaling proteins expressed in Beas2B cells.

(G) Western blotting upon expression in 293T cells of EML4-ALK variants or oncogenic full-length ALK (F1174L) found in neuroblastoma (NB). n = 4.

(H) Model for membraneless cytoplasmic protein granule-based oncogenic RTK/RAS/MAPK signaling. KD denotes the kinase domain of the RTK fusion oncoprotein.

For (A), (B), (D), and (E), starred proteins both co-precipitate and locally enrich by imaging, non-starred proteins only enrich at granules by imaging. WCL denotes whole cell lysates. Images representative of at least 4 replicates. For (C) and (F), arrows indicate a representative EML4-ALK or CCDC6-RET protein granule with local enrichment of respective signaling proteins (multiple non-highlighted granules also show colocalization). All listed signaling proteins showed greater than 85% colocalization with EML4-ALK or CCDC6-RET granules. n = 3.

See also Figure S7.