Rabbit anti pgc 1α

Total proteins were extracted in each group using RIPA buffer (CellNest, Minato, Tokoy, Japan) with Protease Inhibitor Cocktail Set III (1:1000, Millipore, Billerica, MA, USA). Protein concentration was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Protein samples were separated by SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane, blocked with 5% skim milk in Tris-buffered saline (TBS), and probed using various antibodies. The Western blots were visualized using ECL (Bio-rad) and exposed to Amersham™ Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden). The equivalence of protein loading was verified by probing for actin. The antibodies used were as follows: mouse anti-Mfn2 (1:500, Abcam); mouse anti-HO-1 (1:500, Abcam); rabbit anti-PGC1α (1:1000, Abcam); rabbit anti-iNOS (1:1000; Cell Signaling); rabbit anti-Nrf2 (1:1000, Abcam); rabbit anti-pink1 (1:500, Novus Biologicals, CO, USA); mouse anti-parkin (1:1000, Cell Signaling); mouse anti-β-actin (1:1000, Santa Cruz); horseradish peroxidase-conjugated anti-rabbit or -mouse antibodies (1:2500, Abcam).

The expression levels of GPR39, SIRT1, PGC-1α and Nrf2 were measured at 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days post-HIE by Western blot following the manufacturer’s recommendations [34 (link), 35 (link)]. To analyze whether GPR39 receptor and SIRT1/PGC-1α/Nrf2 pathway were involved in the neuroprotective effects of TC-G 1008, the expression levels of GPR39, SIRT1, PGC-1α and Nrf2, and pivotal inflammatory cytokines IL-6, IL-1β, TNF-α were assessed via Western blot. RIPA lysis buffer (Santa Cruz Biotechnology, USA) was used to obtain whole cell lysates. Primary antibodies used were rabbit anti-GPR39 (1:500, Bioss), mouse anti-SIRT1 (1:2000, Abcam), rabbit anti-PGC-1α (1:1000, Abcam), rabbit anti-Nrf2 (1:1000, Abcam), rabbit anti-interleukin (IL)-1β (1:1000, Abcam), rabbit anti-interleukin (IL)-6 (1:1000, Abcam), mouse anti-TNF-α(1:500, Abcam) and mouse anti-β-actin(1:3000, Santa Cruz). The next day, the anti-rabbit (or anti-mouse) secondary antibodies (1:3000, Santa Cruz Biotechnology, USA) were incubated at room temperature for 1–2 h. The gray values were quantified and analyzed by Image J software (NIH).

The total protein was extracted from primary-cultured hippocampal neuronal cells and rat brain tissues using the total-protein extraction kit with Protease Inhibitor Cocktail (Thermo Fisher, Waltham MA, United States). Protein samples containing 60 μg proteins were separated with 8–12% sodium dodecyl sulphate (SDS)-PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica MA, United States). The membranes were then incubated for 1 h with 5% fat-free milk and incubated with the primary antibodies, rabbit anti-SIRT1 (1:200, Biorbyt), rabbit anti-FOXO1 (1:4,000, Abcam), rabbit anti-PGC1α (1:1,000, Abcam), rabbit anti-Bax (1:1,000, Abcam), rabbit anti-Bcl-2 (1:500, Abcam), and rabbit anti-GAPDH (1:10,000, Abcam), overnight at 4°C. Then, the membranes were incubated with appropriate secondary antibodies (1:5,000, Thermo) for 1 h at room temperature. Ultimately, the blots were visualized by ECL chemiluminescence and the protein quantification was measured using Image J software. The relative protein levels were normalized with GAPDH.

For immunoblotting, 75 μg of total protein per sample were loaded into each lane for PGC-1α and GLUT4 quantification. Three controls were run simultaneously with each gel: a wide-range molecular weight marker (Bio-Rad) and total protein isolated from adult rat heart and liver. For each gel, at least one sample per group was included and grouped according to muscle type. Total protein was separated on 7.5% gels for 20 min at 80 V followed by 125 V for 1 h and then transferred to PVDF membranes for 1 h at 100 V. Membranes were blocked in 5% non-fat dry milk in PBS-T for 0.5 h then incubated with either a rabbit anti-GLUT-4 (Milipore Inc., Billerica, MA; 1:600) or a rabbit anti-PGC-1α (Abcam, Cambridge, MA; 1:1000) antibody overnight at 4°C. After washing 3 × 10 min in PBS-T, the membranes were incubated in an anti-rabbit IgG-HRP (Jackson Immunology, West Grove, PA; 1:1000) secondary antibody. Membranes were developed using Thermo Scientific Pierce ECL Western Blotting Substrate. Densitometry and quantification were completed using the ChemiDoc MP Imaging system (Bio-Rad) and Image J software (Rasband, 1997–2007 ).