1. BRAF in-frame deletions mainly function as BRAF homodimers.
BRAF in-frame deletions mainly function as BRAF homodimers. A, detection of BRAF/CRAF heterodimers and BRAF homodimers in H2405, BxPC-3, OV-90, and A375 cells ectopically expressing p61BRAFV600E using in situ PLA. B, quantification of in situ PLA signals of A (mean ± SEM). The number of PLA signals per cell with at least 1,000 cells for all reactions in triplicate was quantified and analyzed by Cellomics ArrayScan VTI Reader and HCS software (**, P < 0.01; ***, P < 0.001, one-tailed t test). C, detection of BRAF/CRAF heterodimers and BRAF homodimers in HEK293 cells ectopically expressing BRAFE586K, BRAF L485-P490 deletion (ΔBRAF) with or without R509H, or empty vector (control) using PLA. PLA signals (red spots) were examined under a confocal microscope and representative images were shown from at least two or three independent experiments. D, quantification of in situ PLA signals of C (mean ± SEM) in HEK293 expressing the indicated BRAF proteins. E, detection of BRAF homodimer by IP and Western blot analysis. HEK293 cells stably expressing FLAG-tagged ΔBRAF or ΔBRAF R509H protein were transfected with vectors encoding MYC-tagged ΔBRAF followed by IP using anti-FLAG or anti-MYC antibody–conjugated beads. The input and IP-prepared proteins were subjected to Western blot analysis with anti-FLAG and anti-MYC antibodies. F, CRAF S338 phosphorylation and MAPK activation in ΔBRAF-transfected HEK293 and NIH/3T3 cells.
Shih-Hsun Chen et al. Cancer Discov 2016;6:
©2016 by American Association for Cancer Research