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. 2014 Apr 1;9(4):e92408. doi: 10.1371/journal.pone.0092408

Figure 1. Enhanced interaction of COMMD1 with ALS-associated SOD1 mutant proteins relative to SOD1 wild-type.

Figure 1

A. HEK293T cells were transient transfected with empty vector (EV), HA-COMMD1 alone or in combination with SOD1-GST constructs (WT, A4V, G37R, G85R, D90A, G93A, E100G). GST-proteins were precipitated by means of GSH-sepharose beads prior to detection of their interaction with COMMD1 as visualized by immunoblotting for HA-COMMD1 (GST PD; upper panel), as described previously [27]. 30 μg of protein lysates were used for detection of WT and mutant SOD1-GST and HA-COMMD1 in total cell lysates (Input; lower panel) using antibodies directed against the HA- or GST-fusion proteins. Tubulin was used as loading control. B. Densitometric quantification of interaction strength between COMMD1 and SOD1 WT versus SOD1 mutants (A4V, G37R, G85R, D90A, G93A, E100G; GST PD), normalized for total SOD1 expression (input). Binding of COMMD1 to SOD1 WT was set at 1. * indicates significantly increased binding of COMMD1 to mSOD1 compared to SOD1 WT – COMMD1 (* p<0.05, ** p<0.005, *** p<0.0001). n.s.  =  non-significant. C. HEK293T cells were transient transfected with HA-COMMD1 alone or in combination with SOD1-GST constructs (WT and G93A). Cells were incubated overnight under basal conditions or with 150 μM CuCl2, lysed, and GST fusion proteins were precipitated by means of GSH-sepharose beads prior to detection of their interaction with COMMD1 as visualized by immunoblotting for HA-COMMD1 (GST PD; upper panel). 30 μg of protein lysates were used for detection of SOD1-GST WT and G93A and HA-COMMD1 in total cell lysates (Input; lower panel) using antibodies directed against the HA- or GST-tags.