Fig. 4: In situ LOXs activity assay applied to aortic tissue samples.

a Representative Western blot images of LOXL2, LOX, LOXL1 and LOXL3 expression in aortic matrix of young (Y, < 3 months old) and old (O, > 18 months old) wild-type (WT) and LOXL2+/− littermate mice (n = 8). AOC3 is detected in the cytosolic fraction of aortic tissue homogenates but not in ECM and AOC3 expression level is similar across four groups. GAPDH was used as loading control. Bar graph shows the densitometry analysis of Western blots (mean ± SEM; n = 6; *P < 0.05 by 2-way ANOVA). b Representative confocal Z-stack maximum projection immunofluorescence images show LOXL2 activity in aortic rings from young and old, WT and LOXL2+/− mice. Freshly isolated aortic rings were incubated with 200 µM biotin-hydrazide (BHZ) in DMEM + ITS for 24 h, then fixed and stained for LOXL2 (red), LOXs activity (green), and nuclei (blue). (n = 6; Scale bar = 100 μm). c Representative confocal images of control aortic rings incubated without BHZ. Samples were stained for LOXL2 (red), endogenous biotinylation (green), and nuclei (blue). (n = 6; Scale bar = 100 μm). d Bar graph of LOXs activity calculated as mean gray value of the IF images (mean ± SEM; n = 12, *P < 0.05 by 2-way ANOVA). The overall LOXs activity in each group corresponds positively to the expression levels of LOXs in aortic ECM shown in a).