Figure 7. Cys-OH formation is increased in ischemic tissues in a mouse hindlimb ischemia model.

A, Representative laser Doppler images of hindlimb ischemia model. Arrows indicate a low perfusion signal (dark blue) detected at immediately after operation (day 0) and a high perfusion signal (yellow to red) detected on day 3 in the ischemic hindlimbs. B, DCP-Bio1 was injected into tibialis anterior (TA) muscles of both ischemic and non-ischemic legs at 3 days after femoral artery ligation. The frozen sections of middle part of TA muscles were incubated with Avidin/Biotin Complex (ABC) reagent and visualized by diaminobenzidine (DAB). Upper left: representative images for DCP-Bio1 positive staining in small vessels (arrows) and skeletal myocytes in TA muscles. Upper right: Bar graph represents the numbers of DCP-Bio1-positive small vessels with different diameters, expressed as percentage of total vessel numbers in TA muscles of 5 randomly selected high power field (×400, per mm²). Lower left: the ischemic tissue sections without ABC reagent showing endogenous biotin used as a negative control. Lower right: DCP-Bio1 staining is not observed in TA muscle from the non-ischemic legs. Bars represent 20 μm. C, Lysates from ischemic tissue labeled with DCP-Bio1 were pulled down with Streptavidin beads, followed by immunoblotting with anti-IQGAP1 antibody to detect biotin bound Cys-OH formed IQGAP1. In parallel, total lysates without biotin pull-down were blotted for IQGAP1 or ERK1/2 (loading control). Bar graph represents averaged data for Cys-OH formed IQGAP1 proteins in response to hindlimb ischemia, expressed as fold change over day 0 (n=3). *p <0.05.