Fig. 3

Orthologous murine donut mutation, I776R, impairs HGF signaling and receptor trafficking.

(A) Immunoblots of Met protein prepared from lysates of TOV112D cells transfected with murine Met^WT or Met^I776R. Upper panels blotted with anti-pY show diminished phosphorylation at tyrosines critical for signaling and lower panels blotted with anti-Met show loading controls. Precursor (white arrows) and mature (black arrows) forms of Met^WT are both detected, but Met^I776R is only present in the precursor form. (B) Quantification by densitometry of phosphorylated Met versus total Met in panel A (t test, * p<0,05; **p<0,01). (C–H) Immunofluorescence staining of HEK293 cells transfected with Met^WT (C,F), Met^I776R (D,G), or furin cleavage-incompetent Met^R306A (E,H). Unfixed, unpermeabilized cells were stained with anti-Met ab1, which binds an extracellular epitope (C–E), and fixed, permeabilized cells were stained with ab2, which binds an intracellular epitope (F–H). Met^WT and Met^R306A are localized to the plasma membrane (C, E, insets in F, H), but Met^I776R is not (D), and is mostly retained in the cytoplasm (G, inset). Intracellular staining of Met demonstrates similar transfection efficiency. DAPI staining (blue) marks nuclei. (I–J) Live imaging of zebrafish blastulae injected with zebrafish Met^WT-mCherry (I) and Met^L775R-mCherry (J) at 5 hpf. Zebrafish Met^L775R-mCherry largely fails to localize to the plasma membrane as compared to Met^WT. Scale bars, 20 μm.