Merlin tumor suppressor function is regulated by PIP2-mediated dimerization
Fig 1
Merlin dimerizes via the FERM domain.
(A). A diagram depicting the dimerization assay using Merlin with either GFP or NanoLuc fused to its C-terminus. (B). A schematic diagram depicting the full-length Merlin (aa 1–595, WT) and the C-terminal deletion mutant (FH, aa 1–511), the FERM domain deletion mutant (HC, aa 315–595) and the dual FERM and CTD deletion (H, aa 315–511). (C). SDS-PAGE gels stained for total protein to access the quantity and purity of isolated probes for Merlin-NL (top) and GFP “bait” proteins (bottom) for GFP, Merlin-GFP, Merlin-FH-GFP, Merlin-HC-GFP and Merlin-H-GFP. (D). Merlin dimerization data quantified on a plate reader and presented as the bound luciferase normalized to 10% of the unbound input NL probe. The data is a mean of triplicate binding reactions with standard deviation and expressed as a percentage of the wild-type Merlin. Inset: an image of the light emitted from Merlin-NanoLuc bound to Merlin and Merlin deletion mutant-GFP bound magnetic beads, performed in triplicate, and dispensed into the wells of a 96-well plate (left). (E). A schematic diagram depicting the N- and C-terminal GFP fused “bait” and the N- and C-terminal fused Merlin-NanoLuc “Probe” constructs used in the BRET assays. (F). Triplicate Merlin dimerization assays for the N-and C-terminal Merlin constructs. The data is a ratio of bound to unbound NL probe, normalized to GFP fluorescence, (mean of triplicate binding reactions with standard deviation and expressed as a percentage of the peak value). (G). Emission spectrum from 400 nm to 600 nm of Merlin dimerization assays normalized to the 450 nm peak. The BRET emission peak at 510 nm is indicated by the arrow.