V3 western workflow complete system

The western blot method was done using V3 Western Workflow™ Complete System, Bio-Rad® Hercules, California, USA). Briefly, 5 mg of striatum tissue was homogenized in Radioimmunoprecipitation assay (RIPA) buffer, then centrifuged at 12,000 rpm for 20 min. The protein concentration for each cell lysate was determined using Bradford assay. Equal amounts of protein (20–30 μg of total protein from cell lysate) were separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride membrane. The membrane was blocked in Tris-buffered saline (TBS) buffer, 3% Bovine serum albumin (BSA) and 0.1% Tween 20 at room temperature for 1 h and incubated with Casp-3, Fas-L and iNOS primary antibodies, supplied by Thermoscientific (Loughborough, UK), overnight at pH 7.6 at 4°C with gentle shaking. After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at room temperature for 1h. Image analysis software was used to read the band intensity of the target proteins against control sample after normalization by β-actin on the Chemi Doc MP imager.

Real‐time PCR was performed for quantitative gene expression of (PPAR α, SREBP1, LC3, ACC1& sirt1) using RNeasy purification reagent (Qiagen, Valencia, CA), reverse transcription reaction (Superscript II, Gibco Life Technologies, Grand Island, NY, USA) and Applied Biosystems with software version 3.1 (StepOneTM, USA). The primers used are shown in Table 1 (Dawood et al. 2018)
Detection of AMPK protein using Western Blot technique (using V3 Western Workflow™ Complete System, Bio‐Rad® Hercules, CA) and antibodies for AMPK and beta‐actin were supplied by Thermoscientific (Rockford, Illinois, USA) (Elattar et al. 2017)