Ubiquitin

Sections were incubated for 1 h in blocking solution containing PBS 0.1% Triton X-100 (v/v) and 5% serum (v/v) and incubated overnight in the primary antibody in 2% blocking solution. The following antibodies were used: ubiquitin (Dako), Nogo-A (R&D Systems), myosin heavy chain subtypes to identify the combination of muscle fibre type (Developmental Studies Hybridoma Bank: myosin heavy chain 2A BF-F3-s, or myosin heavy chain 2B SC-71-s) (see Table S1 for full details of primary antibodies). Primary antibodies were detected using Alexa Fluor 488 or Alexa Fluor 568 secondary antibodies (see Table S1 for full details) and nuclei were stained with DAPI (Sigma-Aldrich). Images were acquired using a Leica DMR fluorescence microscope.
EDL was stained with α-Bungarotoxin (Sigma-Aldrich, T0195) which labels the postsynaptic acetyl choline receptor, and then stained with ChAT antibody (Millipore), a marker of motor axons (see Table S1). Analysis of innervation was performed as follows: percentage of end plates fully innervated; percentage of end plates partially innervated; percentage of end plates completely denervated.

iPS-MNs of each clone were harvested on Day 35 and lysed in RIPA buffer [50 mM Tris-HCl buffer, pH 8.0, 0.15 M NaCl, 1% Nonidet P-40 (NP-40), 0.5% sodium deoxycholate, 0.1% SDS] with protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor (Roche) on ice for 30 min. After sonication with Bioruptor (M2 mode, ON: 20 s, OFF: 20 s, 20 times), samples were centrifuged at 20,000 g for 15 min at 4 °C. The supernatant was used as sample. Each 10- or 15-μg sample was subjected to SDS-PAGE (10-20% polyacrylamide gels, BIO CRAFT, Tokyo, Japan), and separated proteins were transferred to PVDF. The membranes were incubated with primary antibodies, followed by appropriate secondary antibodies, and then visualized using ECL Prime (GE Healthcare, Chicago, IL). The images were acquired on LAS 4000 (GE Healthcare). The following primary antibodies were used in this assay: TFG (1:1,000; Protein Tech), Ubiquitin (1:1,000; DAKO), cleaved caspase-3 (1:1,000; Cell Signaling Technology, Danvers, MA), and β-actin (1:5,000; Sigma-Aldrich).
For detection of cleaved caspase-3, iPS-MNs of each clone were exposed to vehicle or 1 μM MG132 on Day 35 for 24 h. Cells were lysed in RIPA buffer and used for immunoblot analysis with anti-cleaved caspase-3 antibody.

Most chemicals, including MG132, cycloheximide, DMEM, EGF, anti‐FLAG M2 affinity gels, and antibodies to α‐tubulin, FLAG and PIP5Kγ90, were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Lipofectamine 2000, Lipofectamine RNAiMAX, Opti‐MEM I and V5 antibody were purchased from Thermo Fisher Scientific (Waltham, MA, USA). ISA‐2011B was obtained from MedChem Express (Monmouth, NJ, USA). Antibodies to PIP5Kα, β‐actin and green fluorescent protein (GFP) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); NEDD4 (Abcam, Cambridge, MA, USA), ubiquitin (Dako, Carpinteria, CA, USA), HA (Covance, Princeton, NJ, USA), phospho‐Akt (S473) and Akt (Cell Signalling Technology, Danvers, MA, USA) and GST (GE Healthcare, Princeton, NJ, USA) were commercially purchased.