Appropriate secondary antibody

The E64 cysteine protease inhibitor, serine protease phenylmethylsulfonyl fluoride (PMSF) inhibitor, and metalloprotease 1,10-phenanthroline (1,10-PT) inhibitor were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, 38070, France). Fluorescein phalloidin was obtained from Molecular Probes (Thermo Fisher Scientific, 91140 Les Ulis, France). Mouse anti-vinculin antibody (clone hVIN-1) was obtained from Sigma-Aldrich. Appropriate secondary antibodies were purchased from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA, USA) and Molecular Probes Inc. (Thermo Fisher Scientific, Invitrogen Life Technologies, San Diego, CA, USA).

Floating vibratome sections (60 μm) or cryosections (10 μm) were permeabilized using 0.1% Triton X-100 and blocked in blocking solution (PBS, 0.1% Triton X-100, 10% HS; 10% FBS) for 60 min. Antibodies were incubated in blocking solution over night at 4°C. After washing, appropriate secondary antibodies (Jackson ImmunoResearch) were diluted in blocking solution, and incubated for 2 h at room temperature. Slices were mounted onto glass slides using Aqua Polymount (Polysciences). Brains of embryos treated with EdU/IdU were fixed in 2.5% PBS-PFA, cryoprotected in 20% sucrose and cryo-sectioned to 14 μm thick slices before further processing.

For western blot analysis, 30 μg protein was utilized. Blots were probed with specific primary antibodies and the appropriate secondary antibodies (Jackson ImmunoResearch Lab. West Grove, PA). β-actin was used as a loading control. GLUT4 antibody was purchased from Abcam, Cambridge, MA.

Cells were washed twice with cold PBS and lysed by addition of Tris-buffered saline (TBS)-lysis buffer (TBS [50 mM Tris-HCl pH 7.5, 150 mM NaCl], 0.5% Nonidet P-40, 1× protease inhibitor cocktail [Merck], and 1× phosphatase inhibitor cocktail^{42 (link)} followed by incubation on ice for 20 min. Blood cells from healthy control subjects and AML patients were lysed using hot lysis buffer [100 mM Tris-HCl pH 7.5, 5% sodium dodecyl sulfate (SDS)] followed by incubation 5 min at 95 °C and sonication. Some samples were subjected to fractionation using a subcellular protein fractionation kit (Thermo Scientific Pierce), as indicated. Samples were resolved on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were incubated for 1 h at room temperature with blocking solution (0.1% Tween 20/5% non-fat milk in TBS) and then overnight at 4 °C with primary antibodies. Membranes were washed with TBS and incubated for 1 h at room temperature with appropriate secondary antibodies (Jackson ImmunoResearch). Finally, proteins were visualized using a chemiluminescence method (Image-Quant LAS400, GE Healthcare, or ChemiDoc MP imaging system, Bio-Rad). The uncropped scans for all western blot are provided in the Source Data file.

Skin and dorsal root ganglia were post-fixed in 4% paraformaldehyde, cryoprotected in 25% sucrose, embedded in gelatin, sectioned on a sliding microtome and labeled using target-specific antibodies followed by a fluorescently tagged secondary.
Sections were stained with antibodies to keratin K20 (1:20, mouse; Signet Covance, MA), NF145 (1:200, rabbit; Millipore, MA) or PGP9.5 (1:1000, rabbit; Ultraclone, UK) followed by appropriate secondary antibodies (Jackson ImmunoResearch) used at 1:500 dilution. Fluorescent images were captured using a digital camera attached to a Leica DM4000B fluorescence microscope (Leica, Wetzlar, Germany) and processed for brightness and contrast using Adobe Photoshop.

Tissue preparation, staining and analysis was adapted from the protocol from Ippolito and Eroglu (Ippolito and Eroglu, 2010 ). Brains were fixed as above and sectioned at 14 µm. Brain sections were blocked using 20% normal goat serum in PBS for 1 h at room temperature. Vglut1 (1:2500, Millipore, RRID:AB_2301751), PSD95 (1:500, Invitrogen, RRID:AB_2533914), and GFAP (1:1000, Abcam, RRID:AB_296804) were diluted in PBS with 0.3% Triton X-100 and 10% normal goat serum. Cortical sections were incubated with diluted primary antibodies for 42 h at 4°C. Appropriate secondary antibodies (The Jackson Laboratory) were diluted in PBS with 0.3% Triton X-100 and 10% normal goat serum and added to cortical sections for 2 h at room temperature. Slices were mounted using fluoromount-G (Southern Biotech) and imaged with a Nikon A1R confocal microscope. Images were collected with a 100× oil immersion objective and maximum intensity projections (MIPs), of three serial optical sections at 0.175-μm steps, were generated. Five MIPs were generated and analyzed per section. These were averaged to give one value per brain section. Synapses were quantified in ImageJ (RRID:SCR_003070) using the Puncta Analyzer plug in.