Nitrocellulose

The mice were euthanized, and the brains were rapidly extracted and sliced into coronal sections (0.4 mm thick) using a brain matrix followed by either preparation of synaptosomes from the striatum as described above or extraction of tissue from specific brain regions. Tissues from dStr, NAc, ventral tegmental area (VTA), and substantia nigra compacta (SNc) were extracted using a 1.0 mm Harris Uni-Core micropunch (Electron Microscopy Sciences). Synaptosomes and brain tissues were solubilized in the lysis buffer containing 50 mM Tris-HCl pH 7.4, 0.5% sodium deoxycholate, 1% NP-40, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, and phosphatase and protease inhibitors. Lysates were cleared by centrifugation for 15 min at 16,000 × g at 4°C. Aliquots of lysates (30 µg of protein for striatal synaptosomes, dStr, and NAc; 50 µg for whole MB, VTA, and SNc) were denatured in sample buffer at 37°C for 30 min. Lysates were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose (Li-COR), and probed with appropriate primary and secondary antibodies conjugated to far-red fluorescent dyes (IRDye-680 or IRDye-800) followed by detection using Odyssey Li-COR system. Quantifications were performed using ImageJ software.

Samples were separated by 8% or 10% acrylamide gel and stained using InstandBlue (Expedeon) or transferred to Nitrocellulose (0.45 μm, GE Healthcare) or Immobilon‐P PVDF membrane (0.45 μm, Millipore) at 300 mA for 3 h. Membranes were blocked in 5% milk (skim milk powder, LP0031, Oxiod) in 1× PBS (P1379, Sigma‐Aldrich), incubated with primary antibodies diluted in 5% milk/0.1% PBS‐Tween‐20 (PBST) for 1 h, washed 3 × 10 min in 0.1% PBST, incubated with the secondary antibody diluted in 5% milk/0.1% PBST for 30 min and washed 3× in 0.1% PBST. Antibody signals on Nitrocellulose membranes were detected using Odyssey CLx (Li‐COR), and those on PVDF membranes were collected on an Amersham Imager 600 (GE Healthcare).

Log phase cells expressing wildtype or E386V mNeon-DdMyo7 were grown on bacteriological plastic plates (150 mm) were rinsed twice in PB then resuspended, counted and 5 × 10⁷ cells collected by centrifugation (Beckman J6, 200 x g). The pellet was resuspended in 1 ml lysis buffer (100 mM Tris, pH 8.1, 5 mM MgCl₂, 5 mM EDTA and 2.5 mM EGTA), washed once then lysed with Lysis Buffer +1% Tx-100, 1 mM TLCK (Sigma), 1 mM TPCK (Sigma), 1X HALT protease inhibitor cocktail (Pierce ThermoFisher) at room temperature. The 0.5 ml sample was spun immediately at 20,000 x g, 4 °C for 20 min. The supernatant was collected and pellet resuspended and homogenized in 0.25 ml of lysis buffer. An equal volume of ULSB gel sample buffer was added to aliquots from the sup and pellet and the samples run on a 4–15% gradient TGX SDS PAGE gel (BioRad) then transferred to nitrocellulose (Licor). The blot was probed for the presence of DdMyo7 and actin followed by fluorescent secondary antibodies (LiCor) and then imaged with the LiCor Odyssey. Quantification of the DdMyo7 band was performed using Image Studio Lite (Licor).

The cells rinsed with ice-cold HBSS were solubilized in TGH (1%Triton X-100, 10% glycerol, 20 mM HEPES, 50 mM NaCl) lysis buffer supplemented with 1% deoxycholate, and protease (including iodoacetamide and MG132) and phosphatase inhibitors for 30 to 60 min at 4 °C on low speed shaker. Lysates were centrifuged at 16,000 × g for 10 min to remove insoluble material. Aliquots of cell lysates were denatured in Laemmli buffer (2%SDS, 5% 2-mercaptoethanol, 10% Glycerol, 62.5 mM Tris HCl, pH6.8) for 5 min at 95 °C, resolved by 7.5% SDS-PAGE, transferred to nitrocellulose (Li-COR), and probed with appropriate primary and secondary antibodies conjugated to far-red fluorescent dyes (IRDye-680 or -800) followed by detection using an Odyssey Li-COR system. Quantifications were performed using Li-COR and ImageJ software. Molecular mass of stained Precision Plus protein standards was verified by Coomassie Blue staining of unlabeled Precision Plus protein standards from Bio-Rad.