Irdye 800cw goat anti rabbit or goatanti mouse secondary antibodies

Cells were lysed by bead disruption in NP-40 buffer containing cOmplete Protease Inhibitor
Cocktail (Roche, Indianapolis, IN), 100 mM phenylmethylsulfonyl fluoride, and 0.5 M benzamidine.
Proteins were immunoprecipitated with 2 μg of Byr4 antiserum raised against full-length
protein (Cocalico Biologicals, Reamstown, PA) under denatured conditions (NP-40 containing SDS;
Gould et al., 1991 (link)). For gel
mobility shifts, immunoprecipitates were washed in phosphatase buffer (25 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid–NaOH, pH 7.4, and 150 mM NaCl) and
incubated at 30ºC for 30 min with or without λ-phosphatase (NEB). Proteins
were separated on 6% Tris-glycine gels containing 2 μM Phos-tag and 40 μM
MnCl₂ (Kinoshita et al.,
2009) and transferred to PVDF membranes by electroblotting. After incubation of membranes
with polyclonal rabbit anti-Byr4 (1:5000; Cocalico) or monoclonal mouse anti-PSTAIRE (Sigma-Aldrich,
St. Louis, MO) antibodies and subsequent incubation with IRDye 800CW goat anti-rabbit or goat
anti-mouse secondary antibodies (LI-COR, Lincoln, NE), proteins were visualized by an Odyssey
infrared imaging system (LI-COR).

Total protein from BFP expressing iNeurons was extracted using CelLytic^™ M Cell Lysis Reagent (Sigma-Aldrich, #C2978) with PhosSTOP^™ (Roche, #04906845001) and cOmplete^™ ULTRA Protease Inhibitor Cocktail (Roche, #05892970001). Protein concentrations were determined by Qubit^™ Protein BR Assay kit (Invitrogen, #A50668). Equal amounts of protein were denatured at 99°C for 5 minutes, separated by SDS-PAGE, and transferred onto NC membranes. The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 hour, followed by incubation with primary antibodies at 4°C overnight (Antibodies and dilutions in Table S3). The blots were washed with TBS-T followed by incubation with IRDye 800 CW goat anti-rabbit or goat anti-mouse secondary antibodies (LI-COR Biosciences, 1:8000) for 1 hour. After additional washes with TBS-T, the immune complexes were detected by the Odyssey Imaging Systems (LI-COR Biosciences). Image Studio TM Lite Software (LI-COR Biosciences) was used to quantify the protein signals.