Odyssey blocking buffer

Whole E13.5 embryos were lysed in lysis buffer consisting of 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, supplemented with protease inhibitor cocktail tablet (Roche, Indianapolis, IN). Lysates containing 1 mg of protein were incubated with α-Wdfy3 antibody (Novus, Littleton, CO) overnight and then immunoprecipitated using Dynabeads Protein A Immunoprecipitation Kit (Life technologies, Carlsbad, CA). Subsequently, total Protein lysates, immunoprecipitated, and flow-through samples were electrophoresed in NuPAGE 3-8% Tris-Acetate gels (Invitrogen, Carlsbad, CA). Proteins were transferred to PVDF membranes and blocked in Odyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE). Membranes were incubated with α-Wdfy3primary antibody (1:1000; Novus, Littleton, CO) diluted in Odyssey blocking buffer containing 0.1% Tween-20, overnight at 4°C, washed with 0.1% Tween-20 in PBS, before incubating with Li-Cor IR secondary antibody (1:5,000; Li-Cor Biosciences, Lincoln, NE) in Odyssey blocking buffer washed, and imaged on the Li-Cor Odyssey IR scanner. The 333 kDA Wdfy3 isoform, present in all samples served as loading control.

For Western blot analysis Lcells, NCad-Venus Lcells and hippocampal neurons grown in 6 cm cell culture dishes were washed with PBS and lysed in 1.5 x LDS sample buffer (life technologies) supplemented with DTT (10 x reducing agent, life technologies) and protease inhibitors (Calbiochem). Lysate aliquots equivalent to 5 μg of total protein were loaded on 4 – 12 % BisTris NuPAGE gels (life technologies), separated by electrophoresis with MOPS running buffer (life technologies) and transferred to Immobilon-FL (Millipore) using the TurboBlot system (Biorad) at 25 V, 2.5 A for 7 min. Ponceau stain (Sigma) was used to validate homogeneous loading and transfer. Membranes were blocked for 1 h with Odyssey blocking buffer (Li-cor) and incubated overnight at 4 °C with primary antibodies in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were rinsed several times with water followed by 2 × 10 min washes in PBS containing 0.2 % Tween20. Secondary antibodies coupled to IRdye680 and IRdye800 (Li-cor, 1:15.000) were applied for 1 h in a 1 + 1 mixture of Odyssey blocking buffer and PBS containing 0.2 % Tween20. Membranes were washed for 10 min in PBS containing 0.2 % Tween20 and 0.01 % SDS, 10 min in PBS containing 0.2 % Tween20 and rinsed several times in water before the signal was scanned using an Odyssey Infrared imager (Li-cor).

Axon or soma were lysed in RIPA buffer (Sigma-Aldrich) containing anti-peptidase cocktail (Roche). Lysates were heated in LDS sample buffer and separated on NuPAGE^® Novex^® 4–12% Bis-Tris Gels (ThermoFisher). Proteins were transferred to a Odyssey nitrocellulose membrane (LI-COR Biosciences), and membranes were blocked with Odyssey blocking buffer (LI-COR Biosciences) for 1 h. Proteins were detected by overnight incubation at 4 ºC with antibodies against TH, 1:1000 (Cell Signaling Technology Cat# 13106, RRID:AB_2798122) or β-actin, 1:1000 (Cell Signaling Technology Cat# 12620, RRID: AB_2797972) in Odyssey blocking buffer containing 0.2% Tween 20 for either 2 h at room temperature or overnight at 4 °C, followed by incubation with goat anti-rabbit IgG (H+L) IRDye 800CW (LI-COR,Lincoln, cat# 926–32211, RRID: AB_621843 at a 1:5,000 dilution in Odyssey blocking buffer containing 0.2% Tween 20 and 0.1% SDS for 1 h at room temperature. The membrane was washed 3 times with Tris-buffered saline/0.1% Tween 20 (TBS–T) between each incubation step. The membrane was then imaged with the Odyssey Infrared Imaging System (LI-COR Biosciences).