Anti ptrkb

Control and treated cells were collected and lysated as previously described and the protein amount were evaluated [43 (link), 44 (link)]. 30 μg of proteins were loaded and separated on precast 4–20% gradient Bis-Tris gel in running buffer at 100 mV for 70 min followed by transfer to PVDF membranes using a semi-dry device (Thermo scientific, UK), then blocked in 5% no-fat milk for 30 minutes. Membranes were incubated with the subsequent primary antibodies overnight: anti-p-AKT (1:1000), anti-PI3K (1:1000 Cell Signaling, USA), anti p-CREB (1:500 Cell Signaling, USA), anti-pTrkB (1:2000 Cell Signaling, USA), anti-mBDNF (1:500 Abcam, UK), anti-pJNK (1:1000 Santa Cruz, USA), anti-p75(1:1000 Abcam, UK), anti-pro-BDNF(1:1000 Millipore, USA), anti-pERK5 (1:1000 Cell Signaling, USA), anti p-ERK1,2 (1:1000 Santa Cruz, USA). After different washes, membranes were incubated with 1:10000 horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG. The protein bands were detected, normalized and analyzed to actin (housekeeping). Anti-β-actin (HRP-conjugate) (1:10000) has been used. To reprobe, membranes have been stripped with Restore stripping buffer (Thermo Scientific, UK) following manufacturer’s instructions.

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.