Fig. 4
PROTAC 2 decreased the compactness of oligomeric intermediates C-TDP-43 and reduced the high molecular weight oligomers in Neuro-2a cells. A Schematic illustration of FLIM-FRET analysis on the 2FP-C-TDP-43 oligomeric intermediates in the cytoplasm of Neuro-2a cells with or without PROTAC 2. (2FP-C-TDP-43 represents co-expressing eGFP-C-TDP-43 and mCherry-C-TDP-43.) B The color-coded images of the EFRET distribution throughout Neuro-2a cells (“frame” fitting model, upper panel) and its per-pixel distribution histograms (lower panel). The palette (color coding on the upper-right) corresponded to the EFRET levels of overall C-TDP-43 species (monomers + oligomeric intermediates + aggregates). C, D The average EFRET (C) and the population (D) of C-TDP-43 oligomeric intermediates in 2FP-C-TDP-43 expressed Neuro-2a cells with or without PROTAC 2 (5 μM) by applying “highlighted-pixel” fitting model. Each cell was arbitrarily selected (n = 20) and calculated according to the region average lifetime on a pixel-by-pixel basis. Statistic results were shown as mean ± SD (n ≥ 3). Data were analyzed by two-tailed unpaired t-test (*P < 0.05, ***P < 0.001). E Neuro-2a cells expressing eGFP-C-TDP-43 with or without treatment of PROTAC 2 (5 μM) or MG132 (2 μM) were fractionated by applying FPLC on the size exclusion column (SEC). The elution of proteins was monitored by absorbance at 280 nm and fractions were collected every 1 mL. Fractions 9–14 were further loaded on NC membrane and probed with A11 antibody. The elution times of two standards, 670 kDa and 158 kDa, were marked as arrowheads