Nb600 532

Normal Human Epidermal Keratinocytes were lysed in whole cell extraction buffer (25 mM Tris HCL buffer pH 7.5; 0.25 M saccharose 0.2 mM MgSO4; 20 mM EDTA; 0.4 % Triton X-100; 2 mM DTT; 5 µg/mL leupeptin; 0.4 mM PMSF). CD271 protein levels were determined by capillary electrophoresis immunoassay by following the WES user guide from ProteinSimple and after determination of primary antibody dilution. Cell extracts samples (1.92 µg of protein/lane) were mixed with a 5 × Master Mix (DTT, fluorescence labeled maker, SDS) and then heated at 70 °C for 10 min. The samples, the biotin labeled protein ladder (12 kDa, 40 kDa, 66 kDa, 116 kDa, 180 kDa, and 230 kDa), blocking reagent, CD271 Receptor antibody at a dilution of 1:100 (NBP2-19669, Novus Biologicals Europe), HRP-conjugated secondary antibody, luminol S/peroxide, and separation and stacking matrices were also dispensed to designated wells plate. After plate loading, the electrophoresis and immunodetection steps took place in the capillary system (ProteinSimple WES, Santa Clare, CA, USA) and were fully automated with instrument default settings. Peak areas were determined using Compass software (Protein Simple) and normalized to β-actin (loading control) (NB600-532, Novus Biologicals Europe).

The antibodies used in this study were NANOG (R&D Systems, AF1997, 1:500), OCT4 (STEMCELL Technologies, 60093, 1:1000), SOX1 (STEMCELL Technologies, 60095, 1:1000), SOX17 (R&D Systems, AF1924, 1:500), DESMIN (Invitrogen, PA5–16705, 1:500), MAP2 (Proteintech, 17490–1-AP, 1:500), TUJ1 (Neuromics, CH23005, 1:500), GFAP (STEMCELL Technologies, 60128, 1:500), TBR1 (Abcam, ab31940, 1:500), FOXG1 (Abcam, ab18259, 1:500), SYNAPSIN (EMD Millipore, 5747777, 1:500), VGLUT1 (Synaptic Systems, 135311, 1:500), GAD 65/67 (Sigma-Aldrich, G5163, 1:500), PGRN (R&D Systems, AF2420, 1:1000) (for immunofluorescence); PGRN (Sigma-Aldrich, HPA008763, 1:1000), CTSD (R&D Systems, AF1014, 1:1000), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for conventional western blot); V5 (Abcam, ab27671, 1:500), ACTIN (Novus Biologicals, NB600–532, 1:10,000) (for automated WES western blot) (Table S1).

The antibodies used in this study were anti-C1q (Abcam, ab182451, 1:500), anti-DppII (R&D Systems, AF3436, 1:500), anti-Foxp2 (Abcam, ab16046, 1:500), anti-Gfap (STEM CELL Technologies, 60128, 1:500), anti-Iba1 (WAKO, 019-19741, 1:500), anti-Lamp1 (BD Biosciences, 553792, 1:500), anti-Pgrn (R&D Systems, AF2420, 1:1000), TDP-43 (Proteintech, 10782-2-AP, 1:500), anti-Tuj1 (Neuromics, CH23005, 1:500), anti-Vgat (Synaptic Systems, 131011, 1:300), donkey anti-goat IgG (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-11006), donkey anti-rabbit IgG (H + L) Alexa Fluor® 647 (Thermo Fisher Scientific, A-31573), goat anti-rat IgG (H + L) Alexa Fluor® 647 (Thermo Fisher Scientific, A-12247), donkey anti-mouse IgG (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-21202), goat anti-mouse IgY (H + L) Alexa Fluor® 488 (Thermo Fisher Scientific, A-21449) (for immunofluorescence); Ctsd (R&D Systems, AF1029, 1:500), Lamp1 (BD Biosciences, 553792, 1:500), LC3-I/II (Cell Signaling, 2775, 1:1000), Pgrn (R&D Systems, AF2557, 1:100), TDP-43 (Proteintech, 10782-2-AP, 1:1000), p-TDP-43 (Cosmo Bio USA, CAC-TIP-PTD-P03, 1:500), Actin (Novus Biologicals, NB600-532, 1:10,000), donkey anti-goat IgG-HRP (R&D Systems, HAF109), donkey anti-sheep IgG-HRP (R&D Systems, HAF016), donkey anti-rat IgG-HRP (R&D Systems, HAF005), goat anti-rabbit IgG-HRP (for western blot).