Extended Data Fig. 2: Functional analysis of P4B in cells and in vitro.
From: Functional characterization of a PROTAC directed against BRAF mutant V600E
a, P4B dose response analysis in A375 cells. Cells were treated for 24 h with P4B, PEG4-BI, or pomalidomide at the indicated concentrations. p27 accumulation is reflective of G1 cell cycle arrest upon BRAF degradation by P4B treatment. b, Immunoblot analysis of BRAF, CRAF, ARAF, KSR1, SRMS, pMEK and MEK levels in A375 cells after 24 h treatment cells with P4B, P4BME and pomalidomide at the indicated concentrations. c, Immunoblot analysis of BRAF, CRAF, pMEK and MEK levels in SKMEL-28 cells after 24 h treatment with P4B, P4BME and Pmd at the indicated concentrations. Tubulin served as a loading control. d, Time course analysis. A375 cells were treated with 100 nM P4B for the indicated time. e, Anti-BRAF immunoblot of in vitro ubiquitinatated full-length BRAF(V600E). Ubiquitination reactions were carried out + /- the indicated components. The white signal at the bottom of blot corresponds to saturation of BRAF signal. Data shown are representative of minimally two independent experiments.
