West pico luminol

OxyBlot kit (Merck Millipore, Burlington, MA, USA) was performed according to the manufacturer’s instructions to evaluate oxidized protein levels. In brief, cells were plated (density 1.5 × 10⁴ cells/cm²) in 10% FBS-supplemented medium. 24 h after seeding, the cells were exposed to our differentiation conditions, as previously mentioned. After complete differentiation, cells were exposed for 24 h to co-treatment with GSK0660 0.2µM, and 25µM 6-OHDA in culture media supplemented with FBS 1%, as previously described. Consequently, pellets were collected and homogenized in lysis buffer having dithiothreitol (DTT) 50mM. 5 µg/µl of protein extracts were derivatized following manufacturer’s protocols. 20 µg total proteins per sample were separated on 10% polyacrylamide SDS denaturing gels. As primary antibody rabbit anti-DNP diluted 1:150 was incubated. According to manufacturer’s instructions, after incubation with secondary HRP-conjugated anti-rabbit IgG antibody diluted 1:300, the immunoreactive bands were detected with West Pico luminol (Thermo Scientific). Bands were visualized using Alliance Q9 (Uvitec, UK) and analyzed by FIJI software. As housekeeping protein actin was used, and values were given as relative units (R.U.).

Under stereomicroscope, substantia nigra and striatum were isolated and the different regions were freshly lysate using pestles, protein extracted were dosed as previously described [43 (link)]. Tissues lysates containing 10μg of protein were separated on 4–13% gradient Bis-Tris gel in running buffer at 100 mV for 80 min. Proteins were transferred into PVDF membranes using a semi-dry device (Thermo scientific, UK). Membranes were washed in tris-buffered saline with 0.05% Tween20, and blocked in 5% no-fat milk for 1 h at RT. Membranes were then incubated overnight at 4°C with the following primary antibodies, diluted in the same blocking solution: anti p-NRF2 (1:5000 Abcam, UK), anti-NFKB (1:2000 Abcam, UK) anti-p-TRKB (1:2000 Cell Signaling), anti-BDNF (1:500 Abcam, UK), anti-PPARγ (1:500, Thermo, USA) anti-HO1(1:1000 Santa Cruz, USA) at 4°C overnight and then incubated with 1:10000 HRP-conjugated anti-rabbit IgG or anti-mouse IgG. Protein bands were detected with West Pico luminol (Thermo scientific) following kit’s datasheet. Through Alliance Q9 (Uvitec, Cambridge, UK) image chemiluminescent bands were detected and using ImageJ program we analyzed each band intensity normalized as indicated in the “Wester Blotting” in vitro section.

Under stereomicroscope, substantia nigra and striatum were isolated and lysate using pestles to extract and quantify proteins as reported by [30 ]. 20 µg of proteins were run on 4–13% Nupage Bis-Tris precast gel (Thermo Scientific, USA) in running buffer at 200 V for 1 h, then transferred onto PVDF membranes using a semi-dry device (Thermo Scientific, USA) for 10 min in 1 Step buffer. Membranes were incubated with Blocking Buffer (Thermo Scientific, USA) for 15 min at RT and then incubated O/N at 4 °C with the following primary antibodies: anti-TH (1:1000), anti-pCreb (Ser133) (1:500), anti pAKT (Ser473) (1:1000) and anti-mBNDF (1:1000), diluted in the same blocking solution: at 4 °C O/N and then incubated with 1:10000 HRP-conjugated secondary antibody. Protein bands were detected with West Pico luminol (Thermo scientific) using Alliance Q9 (Uvitec, UK). For the densitometric analyses, ImageJ was used and normalized upon the housekeeping protein (HRP-conjugated actin).