Figure 4.

Osmotic stress induces rapid ubiquitylation of TDP-43. A, HEK293E cells were exposed to osmotic shock with sorbitol for 5–60 min followed by fixation and immunostaining of endogenous TDP-43 (green) and the SG marker eIF3η (red) with specific antibodies. Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 20 μm. B, FLAG-TDP-43 and His₆-ubiquitin (+) or empty control vectors (−) were expressed in HEK293E cells that were stressed with 0.4 m sorbitol for 5–60 min. Cells were lysed with 8 m urea lysis buffer, and His₆-ubiquitin–conjugated proteins were isolated with Ni-NTA–affinity purification. Total protein (Input) and eluates (His₆ Pulldown) were analyzed by Western blotting with antibodies against TDP-43, FLAG- and His₆-tagged proteins, ubiquitin, phospho-eIF2α, and GAPDH as loading control.