Dye binding protein assay

We have optimized an ELISA cAMP assay (GE Healthcare) to determine cAMP intracellular levels in dystonia cell models. Briefly, neuron cells were seeded in a 4×10⁴ cells/well (neurons) and 5×10³ cells/well (fibroblasts) in 96-well plates. Fibrolast cells were incubated overnight before the assay, and neurons were culture for 10 days prior to the assay. After 30 minutes incubation with or without 10 µM forskolin, the cAMP levels were determined according to the manufacture's instructions. At least three independent experiments were performed for each assay. Data were normalized to the amount of protein in each sample, as determined using a dye-binding protein assay (Bio-Rad).

Ras-related C3 botulinum toxin substrate 1/Cell division control protein 42 homolog activator (#CN02-A, Cytoskeleton, Inc., San Diego, CA, United States), Antibodies used for immunoblotting and immunofluorescence were as follows: P-Cav-1 (#3251), T-Cav-1 (#3267), P-Src (#2101), T-Src (#2108), and GAPDH (#5274) were all from Cell Signaling Technology (San Diego, CA, United States). P-cofilin (sc-271921) was from Santa Cruz Biotech (Santa Cruz, CA, United States), microtubule-associated protein 2 (MAP2) (ab5392) was from Abcam (Cambridge, MA, United States), anti-neurofilament SMI 31 (#801601) was from BioLegend (San Diego, CA, United States). Primary antibodies were visualized using secondary antibodies conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotech, Santa Cruz, CA, United States) and lumigen ECL ultra (MA-100, Lumigen Inc., Southfield, MI, United States). All displayed bands were compared to molecular weight standards (sc-2035, Santa Cruz Biotech, Santa Cruz, CA, United States). The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad, Hercules, PA, United States). For immunofluorescence, FITC-488, Texas Red-595 and Cy5-647 secondary antibodies were obtained from Molecular Probes (Carlsbad, CA, United States).

Cells were homogenized with cold RIPA lysis buffer followed by 3 cycles of 20-s bursts of sonication at 4°C. The amount of protein per sample was determined using a dye-binding protein assay (Bio-Rad). Electrophoresis was performed on the samples using 4–12% or 10% acrylamide gels (Invitrogen) and transferred to polyvinylidene difluoride membranes (Millipore) by electroelution. Membranes were blocked in blocking solution [20 mM TBS Tween (1%) (TBST) containing 3% bovine serum albumin (BSA)] and then incubated with primary antibodies overnight at 4°C. After 3 washes with TBST, the membrane was incubated with secondary antibody for 1 h at room temperature (RT). Horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (Santa Cruz Biotech) was used as secondary antibody. After another 3 times wash with TBST to remove the rest secondary antibody, membrane was incubated with ECL reagent (Amersham Pharmacia Biotech, Piscataway, NJ, United States) and prepared for imaging. Bands were compared to molecular weight standards (Santa Cruz Biotech). GAPDH served as the internal reference by which to standardize the other protein bands. The calculated ratio of the control group was normalized to 100%, and the comparisons of other groups to the control group were represented as percentages.