Versadoc 5000mp

Cells were lysed with Sample Buffer Solution with Reducing Reagent (6x) for SDS-PAGE (NAKALAI TESQUE, Inc. Cat. No. 09499–14), then the lysates were subjected to SDS-PAGE. Following electrophoresis, proteins were transferred to PVDF membranes (Merck Millipore Ltd. Cat. No. IPVH00010). The membranes were treated with specific primary antibodies reacting to phospho-Marcks, Pkcα, c-Jun, and c-Fos (Cell Signaling TCCHNOLOGY, Cat. No. 2741, 2056, 9165 and 4384, respectively), and actin (SIGMA-ALDRICH, Cat. No. A5060), followed by incubation with ECL™ Anti-Rabbit IgG and treatment with a horseradish peroxidase linked whole antibody (GE Healthcare UK Limited Cat. No. NA934V). Immuno-reactive bands were visualized using ECL™ Prime Western Blotting Detection Regents (GE Healthcare. Cat. No. RPN2232) and the intensity of chemi-luminescent bands was quantitated with Versa Doc 5000MP (Bio-Rad Laboratories, Inc.).

Rat anti-DAT (MAB369; Millipore; 1:2000 dilution) or rabbit monoclonal anti-DAT (AB184451, Abcam, 1:1000 dilution for Fig. S2, A and B only), rabbit anti-TH (AB152, Millipore, 1:10,000 dilution), rabbit anti-pSer40 TH (AB5935, Millipore, 1:5000 dilution), mouse antitransferrin receptor (clone H68.4, Thermo Fisher; 13-6800). Secondary antibodies conjugated to horseradish peroxidase were all from Jackson ImmunoResearch, and immunoreactive bands were visualized by chemiluminescence using SuperSignal West Dura (Thermo Scientific). Nonsaturating immunoreactive bands were detected using either a VersaDoc 5000MP or a ChemiDoc imaging station (Bio-Rad) and were quantified using Quantity One software (Bio-Rad). Surface DAT was calculated by normalizing the biotinylated DAT signal to its corresponding amount of total DAT in that sample, detected in parallel from the same exposure of the same immunoblot. Raw surface DAT values are expressed as %total. For most experiments, DAT surface values following drug treatment were normalized to vehicle-treated samples, obtained from the contralateral hemislice in parallel. Surface DAT bands, and their corresponding total lysate, shown for each experiment were taken from the same exposure of the same immunoblot, and brightness/contrast levels were set identically for all blots. Boxed bands were cropped and rearranged for presentation purposes only.

Protein was harvested using radioimmunoprecipitation assay buffer (Pierce, ThermoScientific, Pierce, Rockford, IL, USA) and 1× protease inhibitor using standard techniques and quantified using DC Protein Assay (BioRad Laboratories, Hercules, CA, USA). Protein extracts were separated on a 4–12% Bis-Tris Gel with 2-(N-morpholino)ethanesulfonic acid (Invitrogen, Carlsbad, CA, USA) and transferred to Immobilon 0.45-µm polyvinylidenefluoride transfer membranes (Millipore, Bedford, MA, USA). Primary antibodies to hSNCA (1:1,000; S3062; Sigma or 1:2,000; MAB5320; Millipore) and β-actin (1:10,000; A5441; Sigma, St. Louis, MO, USA) were used. Blots were developed using electrochemiluminescence Prime Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK) and quantified by VersaDoc 5000 MP (BioRad Laboratories). All samples were compared via t-tests.

After protein was eluted by Mammalian Protein Extraction Reagent and a protease inhibitor (Thermo scientific, IL, USA), protein samples (10 μg/well) were separated by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) and transcribed onto PVDF membranes (Millipore, MA, USA). After the blocking process, they were probed overnight at 4° C with primary antibodies, including polyclonal anti-ZO1 antibody (1:1000, Abcam), polyclonal anti-snail antibody (1:1000, Abcam), polyclonal anti-E-cadherin antibody (1:500, Abcam), polyclonal anti-Transforming growth factor-beta (TGFβ) antibody (1:1000, Abcam), polyclonal anit-ZEB1 antibody (1:2000, Bethyl Laboratories, TX, USA), monoclonal anti-Twist antibody (1:500, Abcam). Thereafter, they were incubated with secondary antibodies for 1 hour at room temperature. Protein detection by a detection reagent (GE healthcare, Japan) was observed by VersaDoc 5000MP (Bio-Rad Laboratories, CA, USA). Subsequently, the blots were stripped off and were reprobed with polyclonal anti-GAPDH antibody (1:1000, Cell Signaling). Densitometric analyses were performed using NIH image software. The concentration of each band was evaluated by the ratio to the GAPDH band in three separate experiments.

C2C12 cells were lysed in 10 mmol/L Tris-HCl (pH 7.8) with 1% Nonidet P-40, 0.15 mol/L NaCl, and a protease inhibitor mixture containing EDTA (Roche Applied Science, Penzberg, Germany). Cell lysates (5 μg of protein) were subjected to SDS-PAGE (10% polyacrylamide gel) under a reducing condition. Following electrophoresis, proteins were transferred onto PVDF membranes and incubated overnight at 4 °C with the primary antibodies against smad1, phospho-smad1/5, ERK1/2, phospho-ERK1/2, ERK5, phospho-ERK5, JNK, phospho-JNK, p38 MAPK, phospho-p38 MAPK, AKT, and phosphor-AKT, followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare, Little Chalfont, UK). Immunoreactive bands were visualized by an enhanced chemiluminescence reaction with an ECL Prime Western Blot Detection System (GE Healthcare). Intensity of the chemiluminescent bands was quantitatively analyzed using Versa Doc 5000 MP (Bio-Rad Laboratories, Hercules, CA, USA). The ratio for intensity of a band for phosphorylated protein to that for total protein was calculated.