Precision plus protein dual color standard ladder

Cell pellets were resuspended in 1X NuPAGE LDS Sample Buffer (Life Technologies) with 2.5% β-mercaptoethanol, heated for 5 min at 95°C, and sonicated. Lysates were loaded into a 4–12% NuPAGE Bis-Tris Protein Gel alongside Precision Plus Protein Dual Color Standard ladder (BioRad), electrophoresed, and transferred onto a 0.45 μm nitrocellulose membrane (BioRad). Membranes were blocked for 2 hours with 5% nonfat dried milk in TBS containing 0.1% Tween-20 (TBST). Membranes were then rinsed three times in TBST and incubated with anti-human GAPDH rabbit antibody (1:1000, Cell Signaling Technology, 14C10) or anti-human NEUROD1 rabbit antibody (1:1000, Cell Signaling Technology, D35G2) in 5% BSA in TBST overnight with gentle rocking at 4°C. Following primary antibody incubation, membranes were rinsed three times in TBST and incubated for 40 min with HRP-conjugated anti-rabbit IgG (1:3000, Cell Signaling Technology, #7074). Membranes were then rinsed with TBST three times and incubated for five minutes in Clarity Western ECL Substrate (BioRad). Membranes were then imaged using 60 min exposure time for NEUROD1 and 10 min exposure time for GAPDH with a ChemiDoc-It² (UVP).