Figure 6
From: TDP-43 self-interaction is modulated by redox-active compounds Auranofin, Chelerythrine and Riluzole
Sequential extraction of TDP-43 after auranofin treatment of N2a cells. Sequential extraction of TDP-43 has been performed of untreated N2a cells (lanes 1–3) and cells treated with 0.1% DMSO (lanes 4–6) as a vehicle control or 10 µM auranofin (lanes 7–9). Soluble proteins obtained after ultrasonication in PBS and ultracentrifugation have been probed for TDP-43 (A) and Hsp90 (D) as cytoplasmic reference protein. Luminal and membrane-associated proteins were extracted in 1% Triton X-100 containing PBS buffer, ultracentrifuged and probed for TDP-43 (B) and protein disulfide isomerase (E) as a luminal ER reference protein. Protein aggregates, cytoskeletal and nuclear proteins were solubilized by 8 M urea in PBS buffer and probed for TDP-43 (C) and Lamin A/C (F) as nuclear matrix protein. There is a strong reduction of TDP-43 extractable with Triton X-100 or 8 M urea after auranofin treatment (compare lanes 7–9 to lanes 4–6 B,H and C,I, respectively) whereas the PBS soluble fraction of auranofin treated N2a cells yields a 34% increase in TDP-43 signal (A,G). Signal intensities were quantified by densitometry on uncropped raw pictures (Supplementary Figure 6) using TINA 2.09 software (Raytest, Germany). Mean and standard deviation are depicted in G,H,I. Error probability was calculated with Student’s T-test, n = 3. For sake of better presentability contrast levels of different extractions were normalized, for uncropped raw data and full-length blots see Supplementary Figure 6.
