Mini protean precast gradient gel

NPCs were cultured for 24 hours with or without 10 µM Y-27632 before being dissociated with Accutase (Innovative cell technologies, San Diego, CA, USA) , pelleted, and lysed in RIPA buffer consisting of 50 mM Tris HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS, 10% NaF, and 1 tablet of protease inhibitor (Sigma, 11836145001). Protein was quantified using the Pierce BCA assay kit (ThermoFisher Scientific #23225) added to 2x Laemmli buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, .004% bromophenol blue, 0.125M Tris HCl, pH 6.8) and boiled for 10 minutes at 95 degrees. For immunoblotting, 20 ug of total cell lysate was resolved by 4–15% Mini-PROTEAN precast gradient gel (Biorad #4561086). Proteins were transferred to a PVDF membrane for 90 minutes at 100V and incubated at room temperature for 2 hours in 3% BSA in TBS-T with antibodies anti-myosin pS19/pS20 (Rockland Immunochemicals, Pottstown, PA, USA; Cat # 600–401-416) and anti-alpha tubulin (Thermo Scientific, Cat #: MS581P1). Blots were visualized with SuperSignal™ West Pico PLUS Chemiluminescent Substrate(ThermoFisher Scientific, #34577) and a Bio-Rad ChemiDoc imaging system.

NPCs were cultured for 24 h with aphidicolin or DMSO vehicle control before being lysed with 2× Laemmli buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl, pH 6.8) and boiled for 10 min at 95°C. For immunoblotting, total cell lysate was resolved by 4–15% Mini-PROTEAN precast gradient gel (Biorad, Hercules, California). Proteins were transferred to a PVDF membrane for 90 min at 100 V and incubated at room temperature for 1 h in blocking buffer (3% Bovine Serum Albumin [Thermo Fisher] in TBS-T [tris-buffered saline 0.1% Tween-20]). The blots were incubated at 4°C overnight in blocking buffer with anti-phosphorylated H2AX and anti-alpha tubulin. Alexa Fluor 800 (mouse) secondary antibodies were used and the blots were visualized on a Licor Odyssey Imaging System (see Table 1 for reagents).