Fig. 2: Quantitative fluorescence imaging measuring the effect of 17-AAG on c-RAF.

a Immunofluorescence images of c-RAF protein in A549 cells treated with DMSO or 17-AAG (1 µM) for 4 h. The upper panels show Hoechst 33342 staining of the DNA (blue) indicating the nuclear region, while the lower panels show the c-RAF immunofluorescence stain. Scale bar −20 μm. b The change in mean c-RAF protein per cell (A549 cells) was calculated for a range of concentrations of 17-AAG and expressed relative to the c-RAF level in DMSO treated samples and the no primary antibody control sample. c Chemical structures of c-RAF inhibitors: BGB-283 (enantiomer), d Novartis pan-RAF inhibitor. e Measurement of the relative c-RAF protein levels in A549 cells treated with a titration BGB-283 or f Novartis pan-RAF inhibitor. c-RAF protein levels are expressed as the fraction of c-RAF present in DMSO treated samples. The interactions between c-RAF and these inhibitors are examples of a small molecule causing a direct effect on protein abundance; c-RAF protein levels are elevated at high concentrations of the small molecule even in the absence of 17-AAG treatment. g Measurement of the relative c-RAF protein levels in A549 cells pre-treated with a range of concentrations (indicated by color) of BGB-283 or h Novartis pan-RAF inhibitor then treated with a titration of 17-AAG (x-axis) for 4 h. c-RAF levels are expressed as fold change in c-RAF levels relative to the DMSO treated sample. Error bars represent standard error of mean (n = 3). Source data are provided as a Source Data file.