Extended Data Fig. 4: CAGE-prox-enabled activation of KRAS and FTO.
From: Time-resolved protein activation by proximal decaging in living systems
a, Experimental validation of all the eight recommended anchor sites of KRAS by ERK phosphorylation upon photo-activation. n = 2. b, Fluorescence imaging of the subcellular localization of wild-type KRAS and KRAS-Y32-ONBY before and after photo-activation, which confirmed correct membrane ___location for these variants inside cells. Scale bars for all frames, 10 μm. n = 2. c, Structural view of the eight computationally recommended anchor sites of KRAS by CAGE-prox. The five positive anchor sites are labelled in orange. d, Schematic of the workflow for dissecting KRAS-mediated intracellular signal transduction. Both the KRAS activation-triggered signal transduction and EGF stimulation-triggered signal transduction were compared by monitoring the downstream phosphorylation responses. e, Upon temporal activation of KRAS, the phosphorylation levels of MEK1, ERK and JNK were analysed by immunoblotting. n = 2. f, Upon temporal stimulation by EGF, the phosphorylation levels of MEK1, ERK and JNK were analysed and compared by immunoblotting. Distinct patterns of downstream kinase phosphorylation were observed with CAGE-prox-enabled temporal KRAS activation as compared to EGF-stimulated samples. n = 2. g, Structural view of the nine computationally recommended anchor sites on FTO by CAGE-prox. h, Incorporation efficiency of ONBY in each of the recommended anchor sites of FTO as measured by immunoblotting. As the incorporation efficiency at the P93 and N205 sites is extremely low, they were excluded from further experimental validations. n = 2. i, The rest of seven FTO variants were validated by the LC–MS/MS-based RNA demethylation assay. Four variants (Y106, Y108, D233 and R322) exhibited desired demethylation activity on m6A upon photo-activation. n = 3. j, The activities of three positive FTO variants (Y106, Y108, and D233) were measured by a substrate demethylation assay. Mean ± s.d.; n = 3; two-tailed t-test. All above-mentioned samples are biological replicates. P values are shown in the figure.
