Fig. 5.

DMT1 can be ubiquitinated and is degraded in response to Co toxicity. (A) Western blot of SH-SY5Y cell line treated with varying concentrations of Co for 18 h shows decreasing DMT1 levels at higher Co concentrations. (B) Control shRNAi SH-SY5Y cells also decrease the level of DMT1 at 400 μM CoCl₂; however Ndfip1 shRNAi cells do not show a significant decrease in DMT1 levels with 400 μM Co. (C) Ndfip1-inducible SH-SY5Y cells when treated with tamoxifen show a decrease in DMT1 protein levels compared to non-tamoxifen treated cells. Addition of the proteasome inhibitor MG132 prevents the decrease in DMT1 levels in the tamoxifen treated cells. (D) Western blot from inducible Ndfip1 SH-SY5Y cells was immunoprecipitated using anti-DMT1 antibodies and probed for polyubiquitin. Induced cells show an increase in DMT1 ubiquitination. Reprobing of the DMT1 IP lanes with anti-DMT1 showed that both lanes contained DMT1 protein.