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. 2006 Feb 13;172(4):497–504. doi: 10.1083/jcb.200505079

Figure 3.

Figure 3.

Association of CRAG with polyQ requires ROS generation. (a) Accumulation of CRAG NI in the brain of an MJD patient. A brain slice (pons) obtained from an MJD patient was immunostained with anti-CRAG antibody. The arrowhead indicates the NI of CRAG. (b) Colocalization of CRAG with polyQ in the NIs. HeLa cells expressing GFP-Q12 or -Q69 are shown as control. HeLa cells coexpressing GFP-Q12/HA-CRAG or GFP-Q69/HA-CRAG were stained with anti-HA antibody (red) and Hoechst 33258 (blue). (c) Association of CRAG with GFP-Q69 but not -Q12. HeLa cells transfected with the indicated constructs were immunoprecipitated with anti-HA antibody (left) or anti-GFP antibody (right) and immunoblotted with anti-GFP or anti-HA antibodies. (d) Association of endogenous CRAG with GFP-Q69 but not -Q12. DRG neuronal cells transfected with the indicated constructs were immunoprecipitated with anti-GFP antibody and immunoblotted with anti-CRAG antibodies and anti-GFP antibodies. (e) ROS scavenger blocked Q69-mediated CRAG activation. COS-7 cells were transfected with the indicated vectors. After 24 h of incubation with or without ROS scavenger Radicut (100 μg/ml), CRAG was immunoprecipitated with anti-HA antibody and an in vitro GTPase assay was performed. (f) ROS scavenger blocked colocalization of CRAG with Q69. COS-7 cells were cotransfected with GFP-Q69 and HA-CRAG in the presence of 100 μg/ml Radicut. After 24 h, an immunofluorescence assay was performed with anti-HA (red) and Hoechst 33258 (blue). (g) ROS scavenger blocked the interaction of CRAG with GFP-Q69. COS-7 cells were cotransfected with GFP-Q69 and HA-CRAG in the presence of the indicated concentration of Radicut. After 24 h, CRAG was immunoprecipitated with anti-HA and immunoblotted with anti-GFP and anti-HA antibodies. Bars, 20 μm.