Figure 2.

Cross-talk between C-terminal ubiquitinylation and phosphorylation of CTF. HEK293E cells were double-transfected with CTF^WT, CTF^4×KR, CTF^K408R, and the phospho-mimic- or -dead CTF^SSDD or CTF^SSAA double mutants (all CTF constructs fused to mCherry) or mCherry-control vector (−). After cell lysis with urea buffer, His₆-ubiquitinylated proteins were affinity-purified with Ni-NTA–agarose. Total protein (Input) and Ni-NTA–agarose eluates (His₆ Pulldown) were subjected to Western blot analysis with antibodies detecting mCherry, TDP-43, ubiquitin, His₆, phospho-TDP-43, and GAPDH as loading control.