Ap106p

All drugs and reagents were from Sigma unless otherwise indicated. The main reagents were as follows: Neurobasal medium (Gibco), B27 Supplement (Gibco), Sulfo-NHS-LC-Biotin (Thermo Scientific), NeutrAvidin Agarose Resins (Thermo Scientific), recombinant human BDNF (R&D System), and K252a (Tocris).
The antibodies used for western blotting were ASIC1a (1:500, sc-13905, Santa Cruz) and GAPDH (1:2000, KC-5G4, KangChen). The HRP-conjugated secondary antibodies used for western blotting were goat anti-rabbit IgG (1:2000, AP132P, Millipore), rabbit anti-mouse IgG (1:2000, AP160P, Millipore), and rabbit anti-goat IgG (1:2000, AP106P, Millipore). The primary antibodies used for immunostaining were GFP (1:1000, A10262, Invitrogen), HA (1:1000, 901501, BioLegend), DsRed (1:1000, 632496, Clontech), and PSD-95 (1:1000, ab13552, Abcam). The secondary antibodies used for immunostaining were donkey anti-human IgG DyLight 550 (1:1000, SA5-10127, ThermoFisher), donkey anti-human IgG DyLight 488 (1:1000, SA5-10126, ThermoFisher), donkey anti-mouse IgG Alexa 647 (1:1000, 715-605-150, Jackson ImmunoResearch), donkey anti-rabbit IgG Alexa 568 (1:1000, A10042, Invitrogen), and goat anti-chicken IgG Alexa 488 (1:1000, A11039, Invitrogen).

ß-Actin (AC14) (abcam, Cambridge, UK; AB6276, 1:20,000 dilution), PARP1 (Proteintech, Rosemont, IL, USA; 66250, 1:650 dilution), PAR/pADR (R&D systems, Minneapolis, MN, USA; 4335-MC-100, 1:1000 dilution), goat anti-mouse (Millipore, Burlington, MA, USA; AP124P, 1:4000 dilution), goat anti-rabbit (Millipore, Burlington, MA, USA; AP156P, 1:10,000 dilution) and rabbit anti-goat (Millipore, AP106P, 1/4000 dilution).

Human myotubes were lysed in 50 mM Tris–HCL, 150 mM NaCl, 10 mM NaF, 10 mM Na₄P₂O₄, 1 mM Na₃VO_4, 1% NP40 and protease inhibitor (Roche, Basel, Switzerland). Protein concentrations were determined using bicinchoninic acid assays. Protein lysates were resolved by SDS-PAGE (8% or 12% acrylamide/bis acrylamide) and were transferred onto a nitrocellulose membrane by wet transfer. Membranes were blocked in 5% (w/v) skim milk in TBST (10 mM Tris–HCL, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) and incubated with primary antibodies to MuSK, LRP-4, anti-Dok7 and Cav3 overnight at 4 °C. Membranes were then incubated with appropriate HRP-conjugated secondary antibodies (HRP anti-mouse [Sigma-Aldrich, Merck-Milllipore 12-348] or HRP anti-rabbit [Sigma-Aldrich, Merck-Millipore AP106P] in 1% (w/v) skim milk in TBST) for 1 h at room temperature. Enhanced chemiluminescence detection was conducted in a mix of Clarity and Clarity Max substrate solution from Bio-Rad (ratio 1:1). Chemiluminescence was imaged on a LI-COR Odyssey, and densitometrical values of bands were quantified arbitrarily using the Image Studio Software (LI-COR Biosciences, Lincoln Neb, USA). Anti-Tubulin was used as the loading control for the normalization of all lanes. Antibody details are listed in Additional file 1: Table S4 (Additional file 1: online resource 2).