0.2 μm nitrocellulose membrane

The cells were lysed in RIPA lysis buffer (Santa Cruz) supplemented with protease inhibitor cocktail (Santa Cruz), then ultrasonicated and incubated at 4 °C for 1 h. The lysates were centrifuged at 12,000 × g for 15 min. Protein concentration was measured using BCA protein assay kit (BioVision). An equal amount of proteins from each sample (50 μg) was separated on 8–12% polyacrylamide-SDS gels and electroblotted onto 0.2-μm nitrocellulose membranes (GE Healthcare). Membranes were blocked using 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated with primary antibodies at 4 °C overnight. Thereafter, the membranes were washed three times with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The primary and secondary antibodies are listed in Supplementary Table 1. Finally, the protein bands on the membranes were visualized by enhanced chemiluminescence (ECL) detection and scanned using an Amersham Imager 680 (GE Healthcare). The integrated density of the bands was quantitatively analyzed using ImageJ software, wherein the band of β-actin was used as an internal loading control.

Samples were resolved by 8 or 10 % SDS-PAGE and transferred to 0.2-μm nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5 % non-fat dry milk in TBST (20 mM Tris, 140 mM NaCl, and 0.1 % Tween, pH 7.6) and probed overnight at 4°C with the following primary antibodies: mouse anti-α-tubulin B512, 1:5000 (Sigma-Aldrich); rabbit anti-mCherry OD78, 1 μg/μL (gift from Arshad Desai); mouse anti-FLAG M2, 1:1000 (Sigma-Aldrich); mouse anti-p150/DCTN1, 1:500 (BD Transduction Laboratories); rabbit anti-ROD/KNTC1 GC7, 1:3500 (in-house); rabbit anti-ROD-1 OD216, 1:5000 (gift from Arshad Desai); rabbit anti-SPDL1/CCDC99 OD157, 1:3000 (gift from Arshad Desai); rabbit anti-ZWILCH, 1:900 (gift from Andrea Musacchio). Membranes were washed three times with TBST, incubated with goat secondary antibodies coupled to HRP (Jackson ImmunoResearch, 1:10000) for 1 hr at room temperature, and washed again three times with TBST. Blots were visualized by chemiluminescence using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) and x-ray film (Fujifilm). Each immunoblot was performed 2 -3 times using samples from independent experiments.

HEK293T cells, primary and iPSC derived neurons were lysed in RIPA buffer (1% NP-40, 0.1% SDS, 150 mM NaCl and 50 mM Tris pH 7.5) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Lysed cells were sonicated for 5 min and clarified by centrifugation at 10,000 g for 10 min at ambient temperature. Protein concentrations were determined using a bicinchoninic acid assay (Thermo Fisher Scientific). Equal amounts of protein (15–30 μg) in 5× Laemmli sample buffer were resolved by SDS-PAGE and transferred to 0.2 μm nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% (w/v) skimmed milk in buffer (150 mM NaCl, 0.1% Tween 20 and 10 mM Tris, pH 7.5), incubated with appropriate primary antibodies overnight at 4 °C, followed by horse radish peroxidase (HRP)-conjugated secondary antibodies (Dako) for 1 h at ambient temperature. Protein bands were visualised using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and Hyperfilm ECL (GE Healthcare) and densitometric analysis was carried out using ImageJ software.

Protein samples were separated on 10% SDS-PAGE and then transferred onto 0.2 μm nitrocellulose membranes (GE Amersham). After being blocked with 5% BSA in TBST buffer containing 0.05% Tween-20, the membranes were probed with the primary antibodies at 4 °C overnight. The membranes were then probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h at room temperature. Protein bands were detected by Prime Western Blotting Detection Reagent (GE life).

PT fractions and the resulting pellets were denatured in 4 × SDS sample buffer (200 mM Tris-HCl, pH 6.8, 10% β-mercapto-ethanol, 8% SDS, 0.08% bromophenol blue, 40% glycerol) by boiling for 10 min. Samples were centrifuged at 14,000 × g for 2 min, at RT and loaded on to SDS-PAGE gels (5% stacking gel, 12% resolving gel) and were blotted onto 0.2 μm nitrocellulose membranes (GE Healthcare, Piscataway, NJ, United States) at 100 V for 1 h. After blocking for 3 h at RT in 5% (w/v) BSA in PBS with 0.05% (v/v) Tween-20 (PBST), membranes were incubated with primary antibodies (Supplementary Table S1) overnight at 4°C. After three washes in PBST for 15 min, membranes were incubated with horse radish peroxidase (HRP) conjugated secondary antibodies (Supplementary Table S1) for 1 h at RT. After rinsing four times in PBST for 20 min, membranes were developed using a chemiluminescence ECL-detection kit (Supersignal West Pico, Pierce, Rockford IL, United States). The molecular weight of proteins of interest were established using a PageRule Plus pre-stained protein ladder, 10–250 kDa (Thermo Scientific).