Gtx101098

Total cell lysates were prepared, and protein concentrations determined with the Bradford assay kit (Pierce Biotechnology, Rockford, IL). Equivalent amounts of proteins were fractionated on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel. Separated proteins were transferred to nitrocellulose membrane (pH 7.9, Amersham Biosciences Inc., Piscataway, NJ), blocked with 5% non-fat powdered milk, and incubated with specific anti-ELF2 (GeneTex; GTX104851), anti-p21 (abcam; ab109520), anti-p27 (abcam; ab32034), anti-c-MYC (GeneTex; GTX103436) and anti-PPARα (GeneTex; GTX101098) primary antibodies at −20°C overnight. After washing, membranes were incubated with HRP-conjugated anti-mouse, anti-rabbit or anti-goat IgG secondary antibody, as appropriate, for 1 h at room temperature. Immune complexes were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham) and Fuji X-ray film.

The following specific antibodies were used for Western blot analysis: anti-UQCRFS1 (1:1000 dilution, ab14746, Abcam), anti-NDUFS1 (1:1000, ab169540, Abcam), anti-VDAC1 (1:3000, ab14734, Abcam), OXPHOS cocktail (1:1000, ab110413, Abcam), anti-LC3 (1:1000, ab51520, Abcam), anti-PINK1 (1:500, ab23707, Abcam), anti-parkin (1:500, #4211, Cell Signaling Technology), anti-ubiquitin (1:1000, P4D1, BioLegend), anti-PGC-1α (1:1000, ab106814, Abcam), anti-PPARα (1:1000, GTX101098, GeneTex), anti-TFAM (1:1000, GTX103231, GeneTex), anti-ATP5A (1:1000, ab14748, Abcam), anti-SDHA (1:1000, GTX101689, GeneTex), anti-SDHB (1:2000, GTX104628, GeneTex), anti-SDHC (1:500, ab155999, Abcam), and anti-SDHD (1:500, ab189945, Abcam) antibodies.

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.