Polyacrylamide gel

Samples (embryos, larvae or caudal fin clips) were lysed in lysis buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.3% Tween-20, 0.3% NP-40, 0.2 mg/mL proteinase K) for 5 h at 55 °C. After inactivation of proteinase K at 95 °C for 15 min, a short fragment including the target site of TALENs or CRISPR/Cas9 was amplified by PCR using the lysates as the templates. The resulting amplicons were electrophoresed on either 15% polyacrylamide gels or 10–20% gradient polyacrylamide gels (Wako Pure Chemical Industries, Ltd.). Supplementary Table S1 lists the PCR primers used.

Cell lysates (10 μg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). After blocking with 4% (w/v) skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% (v/v) Tween 20, membranes were incubated with 1 μg/mL C₄₄Mab−46 or 1 μg/mL anti−β−actin (clone AC−15; Sigma–Aldrich Corp., St. Louis, MO, USA). Membranes were then incubated with peroxidase−conjugated anti−mouse immunoglobulins (diluted 1:1000; Agilent Technologies, Inc.) to detect C₄₄Mab−46 and anti−β−actin. Finally, protein bands were detected with a chemiluminescence reagent, ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca−Imager (DRC Co. Ltd., Tokyo, Japan).

Cell lysates (10 µg) were boiled in sodium dodecyl sulfate (SDS) sample buffer (Nacalai Tesque, Inc.). Proteins were separated on 5–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 4% skim milk (Nacalai Tesque, Inc.) in PBS with 0.05% Tween 20, the membranes were incubated with 10 µg/ml of an anti-CD44 mAb [clone C₄₄Mab-46 (mouse IgG₁, kappa)]; available from Antibody Bank of Tohoku University (ABTU; Miyagi, Japan); http://www.med-tohoku-antibody.com/topics/001_paper_antibody_PDIS.htm#antiCD44) or 1 µg/ml of anti-β-actin (clone AC-15; cat. no. A5441; Sigma-Aldrich Corp.; Merck KGaA). This was followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins (Agilent Technologies Inc.). Finally, protein bands were detected with ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co., Ltd.).

WB studies were performed in 15 patients (11 from Japan and 4 from France). Frozen skeletal muscles were sliced and solubilized in a buffer containing 0.125 M Tris, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, pH 6.8. SDS-PAGE was done following Laemmli’s method. An equal amount of protein (30 μg) was separated on 5–20% polyacrylamide gels (FUJIFILM Wako, Neuss, Germany) and transferred onto a PVDF membrane. The membrane was incubated with the following antibodies against (1:1000 dilution unless otherwise stated): LC3 (1:500) (NB100–2220, Novus Biologicals, Abingdon, UK), GAPDH, (1:1000) (ab8245,Abcam, Cambridge, UK), BNIP3, SQSTM1/p62, beclin- (Abcam, Cambridge, UK), and beclin- 1 (#3738 Cell Signaling Technology, clone (D40C5), MA, USA).

At 48 h after cotransfection of HEK293A cells, the HaloTag-fused protein was labelled with 1 μM diAcFAM ligand (Promega) in FBS-free culture medium for 15 min in a cell culture incubator. Cells were then washed three times with warm PBS and cell lysates prepared in SDS sample buffer at 0, 12 and 24 h after ligand treatment. All lysate samples were separated on polyacrylamide gels (Wako) by SDS-PAGE, and gels were subsequently analysed on a fluorescence scanner (LAS-3000; FUJIFILM, Japan).

Cultured cells were lysed in sodium dodecyl sulfate (SDS) buffer containing 2-mercaptoethanol for Western blotting or in immunoprecipitation assay (RIPA) buffer (20 mM HEPES-KOH [pH 7.4], 125 mM NaCl, 2 mM EDTA, 1% Nonidet-P40, 1% sodium-deoxycholate) containing protease inhibitor cocktail (Roche, Basel, Switzerland) for immunoprecipitation. Ten percent of each cell lysate volume was analysed as the total cell lysate, and the remaining 90% was incubated with anti-FLAG M2 affinity gel (Sigma, St. Louis, MI) or anti-Myc affinity beads (Nacalai) at 4 °C overnight. The affinity beads were subsequently washed with RIPA buffer five times, and the immunoprecipitates were eluted in 2% SDS sampling buffer for 5 min at 95 °C. The eluates were separated on polyacrylamide gels (Wako, Tokyo, Japan) and proteins transferred onto PVDF membranes (Millipore, Billerica, MA). Proteins were detected using an enhanced chemiluminescence system (ECL; Thermo-Fisher Scientific, Waltham, MA or Nacalai). Densitometric analysis of protein bands was performed using ImageJ software^{58 (link)}.

Cell lysates (10 μg) were boiled in sodium dodecyl sulfate sample buffer (Nacalai Tesque, Inc.). The proteins were subjected to electrophoresis on 5%–20% polyacrylamide gels (FUJIFILM Wako Pure Chemical Corporation) and subsequently transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck KGaA, Darmstadt, Germany). After blocking with 4% skim milk (Nacalai Tesque, Inc.), each membrane was incubated with primary mouse mAbs, such as 1 μg/mL of PMab-225, anti-RAP16 tag (PMab-2), or anti-β-actin (AC-15; Sigma-Aldrich Corp., St. Louis, MO, USA), and subsequently with peroxidase-conjugated anti-mouse IgG (1:1000; Agilent Technologies, Santa Clara, CA, USA). Bands were visualized using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) using a Sayaca-Imager (DRC Co. Ltd., Tokyo, Japan).