In vitro binding studies showing ubiquilin-2 proteins carrying ALS mutations have reduced binding with the S5a subunit of the proteasome compared with WT ubiquilin-2 protein.

<p>(A) Left. SDS-PAGE gel stained with Coomassie Blue stained gels showing the different GST purified proteins (right) and the His-tagged S5a subunit of the proteasome that were used in the binding experiments. (B) Equal amounts of GST or GST-ubiquilin-2 proteins were incubated with a fixed amount of His-S5a for 2 hours. After incubation the beads were washed and the amount of His-S5a that was bound to the beads was analyzed by immunoblotting. Equal amounts of the eluate from the binding experiments were separated by SDS-PAGE together with 1/10 of the input S5a-His protein (Lane 1) and either immunoblotted for S5a, or a parallel gel stained with Coomassie Blue. The positions of the different proteins are marked with arrows. The band marked with an * corresponds to BSA that was added as a blocking agent. (C) Quantification of the GST-pulldown results normalized to WT ubiquilin-2 protein. Errors bars show standard error of the mean. The * signifies a statistical reduction in the binding of S5a by several of the GST-fusion ubiquilin-2 mutants compared to ubiquilin-2 WT (p = <0.05).