Quantification of Plasma Apolipoproteins by Western Blot

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.