POLR3 gene and protein expression dynamics in 4H 201 6 Passage 1 – 3 of NES were used for differentiation into neurons using N2 medium, which consisted of DMEM/F-12 (without L-Glutamine, Gibco, 21331020), 1x N2 supplement, 0.1 mM MEM-NEAA, 2 mM L-Glutamine, 2 µg/ml Heparin (Sigma-Aldrich, H3393) and 1x P/S, supplemented with 400 ng/ml Human Sonic Hedgehog (Shh, PeproTech, 100-45) for 4 days. This was followed by 3 days of 10 µM Valproic acid (VPA, Sigma-Aldrich, P4543) in NB+ medium which consisted of NeurobasalTM medium (Gibco, 21103-049) with 1x B27 (Gibco, 17504-044), 18 mM HEPES (Gibco, 15630-056), 0.25x GlutaMAXTM (Gibco, 35050-038) and 1x P/S. On day 8, cells are passaged 1:2 – 1:4 (depending on density) using accutase and maintained in NB+ medium supplemented with 20 ng/ml BDNF (PeproTech, 450-02), 10 ng/ml GDNF (PeproTech, 450-10), 10 ng/ml IGF (PeproTech, 100-11) and 1 µM cyclic-AMP (Sigma-Aldrich, D0260) until day 18. At day 18 the cells were frozen in KSR with 10% DMSO. Protein isolation iPSCs, NES or neurons were quickly thawed in a 37˚C water bath. The cells were resuspended in pre-warmed DMEM/F12 (#21331-020, Technologies) and centrifuged at 300x g for 5 minutes. The cell pellet was washed with 1x Phosphate-buffered saline (PBS, # 14200083, Gibco™) and spun down again. The supernatant was removed and the pellet was resuspended in RIPA lysis buffer consisting of NaCl 150mM (S9888, VWR™), Triton-X100 1% (T8787, Merck), DOC 0,5%, SDS 0,1% (#74255, Merck) and Tris pH 8 50mM (T6066, SigmaAldrich®) freshly supplemented with dithiothreitol 1mM (R0861, Thermo Scientific™), 1x Protease inhibitor (#11836145001, Thermo Scientific™) and 1x phosphatase inhibitor cocktail (#5870s, Cell signaling Technologies™). The cell mixture was put on ice for 30 minutes and centrifuged at 300x g for 20 minutes. The cell lysate was collected in a new tube. The concentration was measured using Bradford reagent (B6916, Sigma-Aldrich®) according to manufacturers protocol. Western blot Invitrogen™ NuPAGE™ LDS Sample Buffer 1X (#NP0007, Thermo Scientific™) was added to protein lysates. The samples were heated for 5 minutes at 100˚C and 20 µg was loaded into a 4–15% Criterion™ TGX Stain-Free™ Protein Gel (#5678084, BioRad) together with the Precision Plus Protein™ All Blue Prestained Protein Standard (#1610373, BioRad). Electrophoresis was performed at 200V for 15 minutes in running buffer (24.8 mmol/L Trisbase, 250 mmol/L glycine, 0.1% SDS). Next, the gel was imaged using the GelDoc EZ Gel Imaging System (BioRad). Following imaging the gel was transferred to an methanol activated (5 minutes) Immun-Blot® PVDF Membrane (#1620177, BioRad) by blotting
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