α synuclein

Fifteen μL of the HSW from each sample was loaded on four gradient SDS-protein gels (Bio-Rad, 4–15%; Hercules, CA, USA) so that a gender- and age-matched patient/control pair was loaded side by side. In total, we analyzed 27 PD samples and 27 gender- and age-matched non-PD samples (14 male and 13 female pairs). The neuroblastoma SH-SY5Y cells were harvested after differentiation with 10 μM retinoic acid for 6 days and lysed with lysis buffer (1% triton X-100 in PBS, 1x protease inhibitor cocktail (Genedepot, Barker, TX, USA)). The SH-SY5Y cell lysates were used as a positive control for the western blot analysis. All samples in the gels were processed for Western blot analysis under the same conditions for each antibody: LRRK2 (MJFF2 Abcam, Cambridge, MA, USA; ab133474, 1 : 1000), α-synuclein (Cell Signalling Technology (CST), Danvers, MA, USA; #2642, 1 : 800), DJ-1 (CST, #2134, 1 : 1000), and TSG101 (Abcam, ab83, 1 : 500). Specific bands of the four separate blots were detected with an enhanced chemiluminescence reagent (WBLUR0500, Millipore, Milford, MA, USA) using the Microchemi 4.2 device (DNR Bioimaging Systems, Jerusalem, Israel) under the same conditions and their densities were analyzed with the Multi-Gauge V 3.0 program (Fuji photo Film, Tokyo, Japan).

The cells were fixed with 4% paraformaldehyde for 5 min at room temperature (RT). After permeabilization with 0.3% Triton X-100 for 10 min and blocking with 5% bovine serum albumin (BSA) for 90 min, the cells were incubated overnight with primary antibodies at 4 °C. Primary antibodies against the following proteins were used for these analyses: MAP2 (chicken, Abcam), TH (mouse, Millipore), cleaved caspase-3 (rabbit, Cell Signaling), LAMP1 (mouse, Santa Cruz), Rab9 (rabbit, Abcam), and α-synuclein (rabbit, Cell Signaling), GPR30 (rabbit, #107748, GeneTex), WASH1 (mouse, SAB4200552, Sigma-Aldrich). The following day, the cells were washed with PBS and incubated with secondary antibodies for 60 min at RT. The secondary antibodies consisted of goat or donkey antibodies conjugated to Alexa 488, 546, 633, or 647 (Invitrogen). Nuclear staining was performed with a Hoechst solution (Invitrogen) containing the secondary antibodies.

Striatal tissue lysates (50 μg protein) and SH-SY5Y cell lysates were separated by SDS-PAGE on 10–12% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were incubated with specific primary antibodies and the antibodies used were DJ-1, Nrf2, phosphoS40-Nrf2 (Abcam 1 : 1000 dilution), Keap1, NQO1, Cullin3, PKC-δ (Pierce Antibodies, 1 : 1000 dilution), γGCLC, HO-1, nitrotyrosine (Santa Cruz Biotech, 1 : 1000 dilution), α-synuclein, and iNOS (Cell Signaling Technology, 1 : 1000 dilution). To verify the uniformity of protein load and transfer efficiency across the test samples, membranes were reprobed with β-actin and Lamin B (Cell Signaling Technology, 1 : 1000 dilution). Immunoreactive bands were developed with Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, Billerica, USA) and visualized by using an enhanced chemiluminescence system (ChemiDoc, Bio-Rad, USA) and presented in comparison to β-actin/Lamin B expression.

We performed Western blotting using previously established experimental methods [36 (link)]. SH-SY5Y cells were lysed, and the supernatant was collected. The total protein concentration was calculated using a Coomassie Plus protein assay kit (Pierce, Rockford, IL, USA). We took 50 µg of cell extract from each sample for electrophoresis analysis of the sodium dodecyl sulfate-polyacrylamide gel and then transferred the separated proteins to a polyvinylidene fluoride membrane. Next, the membrane was blocked and incubated with the primary antibody overnight at 4 °C. The next day, the membrane was washed, and HRP-conjugated secondary antibodies (PerkinElmer Inc., Boston, MA, USA) were added for 1 h at room temperature. Finally, specific protein signals were detected via the ECL substrate and chemiluminescent gel imager. PI3 kinase p100, Beclin-1, Atg 7, LC3, mTOR, phospho-mTOR, caspase 3, cleaved caspase 3, poly ADP ribose polymerase (PARP), cleaved PARP, SIRT1, β-tubulin, α-synuclein, and the β-actin antibody were all purchased from Cell Signaling Technology (Beverly, MA, USA).

Cannabidiol (purity > 98%)were purchased from Biopurify (Chengdu, China). 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyltetrazolium bromide (MTT) and 1-Methyl-4-phenylpyridine (MPP⁺) were purchased from Sigma-Aldrich (St. Louis, MO, United States); Dulbecco’s modified Eagle medium and 10% fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, United States). penicillin and streptomycin were purchased from Sigma-Aldrich (St. Louis, MO, United States). Primary antibodies against TH, SIRT1, α-synuclein, p62, LC3-I/II, Atg5/7, Parkin, PINK-1, Nrf2, SOD-1, DJ-1, GSH, NOTCH1, Hes1, p-p65, p65, p-Ikb, Ikb, and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, United States).