Mouse ha

Following the expression of exogenous proteins overnight, the neurons were fixed in 4% paraformaldehyde in 1X PBS for 5 min and washed three times with 1X PBS for 5 min. The cells were then permeabilized and incubated in a blocking buffer (1% Bovine Serum Albumin, 5% Normal Goat Serum, 0.1% Triton X-100 in 1X PBS) for 30 minutes. Cells were incubated in combinations of the following primary antibodies at the following dilutions: chicken GFP (Millipore, Bellerica, MA, USA), 1∶15000; mouse AnkG (Neuromab, Davis, CA, USA), 1∶500; mouse HA (Covance, Princeton, NJ, USA), 1∶500; rabbit AnkG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1∶1000; rabbit Myosin Va (Sigma, St. Louis, MO, USA), 1∶500; mouse Tubulin (Sigma, St. Louis, MO, USA), 1∶500; rabbit Stau1 (a generous gift from Dr. M. Kiebler), 1∶500 overnight. After incubation with the Alexa fluorophore-conjugated secondary antibodies (all secondary antibodies were used in 1∶2000 dilution (Invitrogen, Carlsbad, CA, USA)) for 30 hours, the neurons were mounted on glass slides using Fluoromount G (Electron Microscopy Sciences, Hatfield, PA, USA).

Cells were processed for immunoprecipitation (IP) and/or SDS-PAGE/Western blotting as previously described in[25 (link)]. Briefly, cells were lysed 48h post-transfection Triton lysis buffer (50mM Tris pH 7.5, 100mM NaCl, 2mM EDTA, 1% Triton X-100, 1X Roche protease inhibitor mix, 1X Roche phosphatase inhibitor mix). Post clarification, HA-Strep Hyd was pulled down using either Streptactin sepharose (GE Healthcare) or HA-agarose (clone HA-7) (Sigma) for 1hr at 4°C with rotation. After washing, protein complexes were eluted with one bead volume of 1X NuPAGE LDS Sample Buffer (Invitrogen) and 100mM DTT. To IP endogenous Hyd, 5μl M19 antibody (Santa Cruz) was added to the lysate and incubated at 4°C with rotation for 2 h, followed by Protein-A agarose (Sigma) for 30 min with rotation. Samples were run on BIS-TRIS-gradient gels (Invitrogen) and blotted onto PVDF (Millipore). Antibodies used were: mouse HA (1:2,000 Covance), FLAG M2 (1:2,000 Sigma), SGG GSK-4G-AS (1:5,000 Euromedex), Myc 9B11 (1:6,000 Cell Signalling), V5 (1:2000 AbD Serotec); goat EDD M19 (1:1,000; Santa Cruz); rat Ci 2A1 (1:10 DSHB); mouse, goat and rat HRP-conjugated secondary antibodies were used 1:5,000 (Jackson ImmunoResearch Laboratories).