Mouse oct3 4

OBs were microdissected from the brain, homogenized, and lysed in RIPA buffer (with Halt, 5 mM EDTA, and DNaseI) on ice. Cell pellets were thawed and lysed in Nonidet P-40 buffer (1% Nonidet P-40 buffer, 50 mM Tris HCl, pH 8.0, 150 mM NaCl) on ice for 45 min. Lysed cells were centrifuged for 20 min at 14,000 rpm in 4°C. The supernate was saved and concentration assessed at 595 nm using a spectrophotometer and the Protein Concentration Assay Reagent (Bio-Rad, Hercules, CA, USA) to generate standards. Twenty micrograms of total protein was loaded into a 10% SDS-PAGE gel and separated for 1 h at 200 V. A polyvinylidene fluoride membrane was activated, and proteins were transferred at 30 V for 1 h at 4°C. Membranes were blocked in 10% milk in 1× PBS for 1 h at room temperature. Primary antibodies were incubated overnight at 4°C and diluted in 5% milk in 1× PBS with Tween 20: mouse GFAP (1:1000; MilliporeSigma), mouse TuJ1 (1:1000; Covance, Princeton, NJ, USA), mouse Oct3/4 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), rat LIN28 (1:500), and mouse β-actin (1:1000; MilliporeSigma). Bound primary antibodies detected using horseradish peroxidase-conjugated anti-mouse (1:10,000) and anti-rat (1:12,500) were incubated for 1 h at room temperature and diluted in 5% milk in 1× PBS Tween 20.