Fig. 4. Cys215 of Insig-2 can be oxidized by ROS.

a Procedure of detecting Cys215 oxidation in Insig-2 using the DCP-Bio1 probe. b The CRL1601 cells were transfected with indicated plasmids and harvested for cysteine sulfenic acid detection as described in the Materials and methods. c C2C12 cells were transfected with a plasmid expressing Insig-2 and then treated with H₂O₂ at indicated concentrations for 10 min. The cells were then harvested for cysteine sulfenic acid detection. d C2C12 cells were transfected with a plasmid expressing Insig-2 and then treated without (−) or with 10 μM H₂O₂ for 5 h. Cells were harvested for immunoblotting analysis. e CRL1601 cells were transfected with plasmids expressing Insig-1 or Insig-2 as indicated and then harvested for cysteine sulfenic acid detection. f Sequence alignment of human and mouse Insig-1 and Insig-2 proteins. Conserved sequences are highlighted in shadow. The YECK tetrapeptide (in red) is exclusively found in Insig-2 but not Insig-1 proteins. Red dot denotes the ubiquitylation site.