Gtx110704

We measured proteins levels by western blotting as described previously (Ge et al., 2007 (link); Li et al., 2017 (link)). To determine plasma levels of ApoA-II, ApoA-I, and ApoE, 0.5 μL samples from each mouse were separated by Tris-Tricine/SDS–16.5% or 15% polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon, 0.2 μm pore, Millipore Corp., MA, USA) and incubated overnight at 4°C with primary antibody solution containing polyclonal rabbit anti-mouse ApoA-II antiserum (diluted 1:3000) or the ApoA-I antiserum (diluted 1:4000) produced in our laboratory, or ApoE antibody (1:500, Santa Cruz, San Francisco, CA, USA). Next, horseradish peroxidase-conjugated anti-rabbit IgG (Code #7074, Cell Signaling Technology Inc, Danvers, MA, USA) (1:3000) was used for 1 hr incubation at room temperature and target proteins were detected by the enhanced chemiluminescence (ECL) method. Thirty micrograms of liver lysates were separated on Tris-Tricine/SDS–12% PAGE to determine levels of PPARa (1:3000, GTX101098, GeneTex Inc), β-actin (1:3000, GTX110564, GeneTex Inc), and catalase (1:3000, GTX110704, GeneTex Inc, CA, USA). Target protein levels were analyzed using the NIH ImageJ software.

We detected AApoAII deposition and catalase by immunohistochemistry (IHC) following a previously described method (Li et al., 2017 (link)). Antiserum against mouse ApoA-II was produced against guanidine hydrochloride-denatured AApoAII in our laboratory (Higuchi et al., 1983 (link)) and applied at a dilution ratio of 1:3000. Catalase antibody was applied (1:500, GTX110704, GeneTex Inc, CA, USA) to reveal the degree of peroxisome change in the liver. After incubation overnight at 4°C with the primary antibody, the sections were incubated with the biotinylated secondary antibody (1:300, DAKO, Glostrup, Denmark) for 1 hr at room temperature. Target proteins were identified by the horseradish peroxidase-labeled streptavidin-biotin method (1:300, DAKO). In the immunofluorescence experiments, the sections were incubated with the PPARα antibody (1:500, GTX101098, GeneTex Inc, CA, USA) overnight and incubated with Alexa Fluor 488 goat anti-rabbit antibody (1:500, Thermo Fisher Scientific, Japan) for 1 hr at room temperature and incubated with DAPI for 10 min. Images were captured immediately using a confocal laser fluorescence microscope (LSM 880 with Airyscan, Carl Zeiss, Germany). In a negative control section, the primary antibody was omitted to confirm the specificity of staining.

The protein levels of the antioxidants copper-zinc superoxide dismutase (CuZnSOD; GTX100554; GeneTex, Irvine, CA), manganese superoxide dismutase (MnSOD; GTX116093; GeneTex), glutathione peroxidase 1 (GPX-1; GTX116040; GeneTex), catalase (GTX110704; GeneTex), and the marker of lipid peroxidation (4-Hydroxynonenal; 4-HNE; ab46545; Abcam, Cambridge, MA) were measured by Western blotting [19 (link)] in isolated liver mitochondria [20 (link), 21 (link)]. The protein content of these blots was normalized to α-tubulin (GTX112141; GeneTex) levels (the loading and transfer control) since alpha-tubulin is an inherent component of mitochondrial membranes [22 (link)]. This method does not address the purity of the mitochondrial isolation, as α-tubulin is not unique to mitochondria; however, centrifugation at 3,500 g during mitochondrial isolation has been shown to minimize contamination by peroxisomes and other organelles [23 ]. A chemiluminescent system was used to visualize marked proteins (GE Healthcare Life Sciences, Pittsburgh, PA). Images were taken with the ChemiDocIt Imaging System (UVP, LLC, Upland, CA).