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. 2016 Jan 11;6:19118. doi: 10.1038/srep19118

Figure 5. CUL2 ubiquitinates CTF35.

Figure 5

(a) In vivo ubiquitination assay of HEK293A cells co-transfected with HA-VHL, Myc-CUL2, and WT and C175S TDP-43-FLAG. TDP-43-FLAG was pulled down with anti-FLAG affinity beads and blotted with anti-Lys48 ubiquitin. A dashed line indicates total ubiquitin species, while a solid line indicates ubiquitinated TDP-43. Ubn indicates polyubiquitin. (b) The same assay showing that CUL2 ubiquitinates CTF35 more than FL TDP-43. Note that double overexpression of VHL and CUL2 reduced the ubiquitinated proteins. A dashed line indicates total ubiquitin species, while a solid line indicates ubiquitinated FL TDP-43 or CTF35. (c) CUL2 knockdown inhibited the ubiquitination of CTF35. At 24 h after the introduction of siRNA for CUL2 (siRNA1 in Fig. 4n), FL or CTF35 TDP-43 was additionally transfected. After further incubation for 48 h, lysates were immunoprecipitated with anti-FLAG affinity beads and analyzed by Western blotting for ubiquitin. A dashed line indicates total ubiquitin species, while a solid line indicates ubiquitinated FL TDP-43 or CTF35.