Ibright cl1000 imaging system

Total RNA from 3T3-L1 and RAW 264.7 cells treated with and without GA was extracted using the RNeasy Plus Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The concentration of extracted RNA was measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized from 50 ng of each RNA sample using SuPrimeScript RT Premix (GeNet Bio, Korea). PCR was performed with equal amounts of synthesized cDNA using Prime Taq Premix (GeNet Bio) and specific primers (Table 1) under the same conditions for RNA samples of both cells (95°C for 1 min, 59°C for 1 min, and 72°C for 1 min; 30 cycles). The PCR products were stained using EcoDye (BioFACT, Korea) and visualized using the iBright CL1000 Imaging System (Invitrogen).

RIPA lysis buffer combined with a protease inhibitor cocktail was used to lyse liver samples (Beyotime Biotechnology, Shanghai, China). The lysates were centrifuged for 20 min at 12,000 rpm at 4 °C, the supernatants were collected, and the protein concentration was measured using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). SDS-PAGE was used to separate the protein lysates, which were then transferred to PVDF membranes. The blotted membranes were blocked for 2 h with 5% skimmed milk, then washed and incubated overnight at 4 °C with antibodies against CYP7A1, HMG-CoA, LDL-R. The bands were seen using an enhanced chemiluminescence reagent and assessed in an iBright CL1000 imaging system after incubation with the relevant primary and secondary antibodies (Invitrogen, Singapore). The expression of β-actin was used as a control for normalizing protein expressions.

Cells and brain tissues were harvested by scraping and lysed in a buffer containing 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM Na₃VO4, 1% sodium deoxycholate, 0.1% SDS, 1 μg/ml pepstatin A, 50 mM NaF, 0.5 mM EDTA, 1 mM EGTA, and Protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein were resolved on an SDS-PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked by 5% skim milk for 1 h at 37 °C and then incubated with primary antibodies against BECN1-regulated autophagy protein 1(AMBRA1), microtubule-associated proteins 1A/1B light chain 3B (LC3B), α-syn, BECN, pAKT, AKT, lysosomal-associated membrane protein 1 (LAMP1), ras-related protein 7 (RAB7), transcription factor EB (TFEB) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma) at 4 °C overnight. Then, the membranes were incubated with the respective secondary antibodies (GeneTex, Irvine, CA, USA) for 2 h at 37 °C. Western blot signals were visualized using the Immobilon ECL Ultra Western HRP Substrate (ECL Plus kit, GE Healthcare, Piscataway, NJ, USA) and captured on an iBright CL1000 Imaging System equipped with iBright Analysis Software (Invitrogen, Carlsbad, CA).