Flag m2

Spinal cords were isolated from control and mutant E18.5 embryos. Tissue lysates were prepared and diluted to protein concentration of 0.4 μg/μl using sample preparation kit (ProteinSimple). Tissue lysates were examined using automated western blot system, WES System (ProteinSimple) as per manufacturer’s protocol. The following primary antibodies were used for analysis: ZPR1 (Clone LG-C61), Flag M2 (#F1804, Sigma), HoxA5, HoxC5, α-tubulin (#T8203, Sigma) and β-Actin (#A5316, Sigma). Data analysis and quantitation of protein levels were performed using Compass Software (Protein Simple). For standard immunoblot method, cell lysates were prepared from control and transfected HeLa cells using Triton lysis buffer^{5 (link)}. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane (Millipore). Proteins were detected using primary antibodies (1:100), ZPR1 (Clone LG-C61), Flag M2 and α-tubulin (#T6074, Sigma) followed by HRP-conjugated donkey anti-mouse or rabbit IgG (1:5000) secondary antibodies. Chemiluminescence and quantitation of immunoblots was performed using ImageQuant LAS4000. The relative levels of proteins (mean ± s.e.m.) normalized to either actin or tubulin, are presented as bar graphs.

Cell lysates (50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton X-100) and freshly added EDTA-free protease inhibitor cocktail (Roche) were immunoblotted with antibodies against PRMT1 (made in-house), MyoD (Santa Cruz), ASYM26 (Millipore), FLAG-M2 (Sigma), and Myc (Sigma). Protein extracts were resolved by SDS-PAGE, transferred to a nitrocellulose membrane using an immunoblot TurboTransfer system (Bio-Rad), blocked for 1 h at room temperature in Tris-buffered saline with Tween 20 (TBST)–5% milk and incubated with primary antibody, followed by incubation of secondary antibodies conjugated to horseradish peroxidase (Sigma-Aldrich). Western Lightning Plus ECL from PerkinElmer was used for chemiluminescence detection.
For immunoprecipitations, 48 to 72 h after transfection, cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl [pH 8.0]) freshly complemented with EDTA-free protease inhibitor cocktail (Roche). Supernatants were collected and incubated with primary antibodies for 1 h on a spinning wheel, and then 25 μl of FLAG-M2 (Sigma) slurry was added and incubated at 4°C for 30 min on a spinning wheel. The beads were then washed 5 times with lysis buffer and boiled with 4× Laemmli buffer prior to immunoblotting.