Protease inhibitor mix

Approximately 500 μg of Triton/Lubrol-solubilized synaptosomes containing 1 mM benzamidine, 5 mM Mg-ATP, 5 mM EDTA, supplemented with protease inhibitor mix (Sigma-Aldrich), in PBS at pH 7.5 was used for each control experiment. In the experimental sets, 1 mg of isolated synaptosomes exposed to 5 μM PA for 1 hr at 4°C was used. Post PA incubation, the synaptosomes were similarly solubilized as control synaptosomes using Triton/Lubrol containing 1 mM benzamidine, 5 mM Mg-ATP, 5 mM EDTA, supplemented with protease inhibitor mix (Sigma-Aldrich), in PBS at pH 7.5. Sucrose density fractionation of solubilized synaptosomes was carried out using a minor modification of a published procedure 20 (link). Two hundred microlitres of the solubilized synaptosome preparation was layered over a sucrose gradient prepared by successively layering 400 μl each of 50%, 40%, 30%, 20% and 10% sucrose. After centrifugation at 240,000 × g in a Beckman Coulter Optima TLX Ultracentrifuge for 3 hrs at 4°C, 200 μl fractions were collected from the top of the gradient designated fractions number 1 through 11. Equal volume of each fraction were resuspended in Laemmli 21 (link), reducing sample preparation buffer and resolved using 10% SDS-PAGE for both Coomassie Blue staining of protein fractions and electrotransfer to nitrocellulose membrane for Western blot analysis.

Subcellular protein fractionation was performed after washing cells twice with ice cold PBS (Sigma). Cell pellets were subsequently lysed in hypotonic cytosol extraction buffer L1 (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% (v/v) NP40, protease inhibitor mix (Sigma, no. P8340) 1:1,000) for 20–40 min on ice. Lysates were collected and centrifuged for 5 min at 2,000×g, 4 °C. Cytosolic extract (CE) supernatants were collected, and nuclear pellets were washed once in L1 and subsequently lysed in RIPA buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP40 (v/v), 1% sodium deoxycholate (w/v), 1 mM EDTA), 1:1,000 protease inhibitor mix (Sigma), 25 U/ml benzonase (Merck Millipore, no. 70746) for generation of nuclear extracts (NE) in a ratio of 5:1 of cytosol to nuclear extract.

Cells expressing Rix1-5FLAG or Nsa1-5FLAG were harvested and washed using STOP buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1mM NaN₃ [pH 8.0]). Cell pellet was mixed with equal amount of buffer 1 (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 1.5 mM MgCl₂, 0.3% NP-40, 1 mM PMSF, 1 mM DTT, 0.1 mM Na₃VO₄, protease inhibitor mix (Sigma)), and the mixture was beaten with glass beads using Fastprep bead-beater. Buffer 2 (50 mM Tris-HCl (pH 7.5), 1.5 mM MgCl₂, 7.5% glycerol, 1 mM PMSF, 1 mM DTT, 0.1 mM Na₃VO₄, protease inhibitor mix (Sigma)) was added in volumes ∼2x the cell pellet, and the mixture was centrifuged (5k rpm, 2 min once, and 14k rpm, 10 min twice) to obtain supernatants (whole cell extracts). Immunoprecipitation was performed by incubating the whole cell extracts with anti-FLAG M2 monoclonal antibody (Sigma) and Dynabeads Protein G (Veritas) for 1 hr at 4°C. Whole cell extracts and immunoprecipitates were subjected to immunoblot analysis using mouse anti-HA monoclonal antibody 12CA5 (abcam), mouse anti-FLAG M2 monoclonal antibody (Sigma), or anti-GFP monoclonal antibody (Roche).