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. 2016 Apr 22;7:11391. doi: 10.1038/ncomms11391

Figure 2. FINDY destabilizes DYRK1A during the folding process.

Figure 2

(a) Schematic diagram of the 2A peptide-mediated bicistronic expression of FLAG-DYRK1A and HA-EGFP. Expression is controlled by the tet operator. Effects of FINDY (b) or RD0392 (d) on destabilization of DYRK1A. FINDY (b) or RD0392 (e) was added with doxycycline to HEK293 cells harbouring the bicistronic expression vector, which was stably integrated in the genome. After incubation for 5 h, total cell lysates were subjected to SDS–PAGE followed by western blot analysis using the corresponding antibodies against FLAG, HA and GAPDH. Representative data from the pentaplicate experiments are shown. (c) The amount of endogenous DYRK1A was decreased by FINDY. Primary cortical neurons were cultured in the presence of FINDY for 3 days. DYRK1A, Tuj1 (neuron-specific class III β-tubulin) and GAPDH were detected by western blot analysis using their corresponding antibodies. Representative data from the triplicate experiments are shown. (e) FINDY did not induce the degradation of mature DYRK1A. The cells were treated with doxycycline for 16 h, then incubated with cycloheximide (CHX) with or without FINDY for the indicated time. Total cell lysates were subjected to SDS–PAGE followed by western blot analysis. Representative data from the triplicate experiments are shown. Conc., concentration.