Sds page pre cast gel

Cells were lysed in RIPA buffer (Pierce) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma). The total cellular extract was separated in 10% SDS-PAGE precast gels (Bio-Rad) and transferred onto nitrocellulose membranes (Millipore). Membranes were first probed with indicated primary antibodies, followed by the corresponding secondary antibodies which were conjugated with horseradish peroxidase (Millipore). Protein detection was conducted using ECL substrate (Pierce) and the signal intensities were quantified by Image-Pro Plus 6.0.

Protein samples were obtained by pooling whole heads of embryos or larvae at 48 or 70 hpf, respectively, in RIPA lysis buffer (89900; ThermoFisher Scientific) containing 1x protease and phosphatase inhibitor cocktail (5872; Cell Signaling Technology, Danvers, MA, USA). Proteins were separated in a 12% SDS‐PAGE pre‐cast gel (4561043; Bio‐Rad Laboratories, Inc.) and transferred to a PVDF membrane (Sigma‐Aldrich Corp., St. Louis, MO, USA). The membrane was incubated in 5% bovine serum albumin (BSA) with 0.05% Tween‐20 for 2 h to block non‐specific binding of the antibodies and then incubated overnight at 4C with rabbit anti‐NeuroD antibodies (Ochocinska and Hitchcock, 2009) diluted 1:1,000 in 2.5% blocking solution. Blots were rinsed with TBS with 0.05% Tween‐20 and incubated with horseradish peroxidase‐conjugated secondary IgG (1:2,000) for 1 h at room temperature. Bands were visualized using the enhanced chemiluminescence assay detection system (34075; ThermoFisher Scientific). For loading controls, blots were stripped in the stripping buffer (21059; ThermoFisher Scientific) for 5 min, processed as described above, and labeled with mouse anti‐βactin antibodies (1:5,000) (NB10074340T, Novus Biologicals, LLC, Littleton, CO, USA). Images were captured using the FluorChem E Imaging System (Bio‐Techne, Minneapolis, MN, USA) and band intensity was quantified relative to βactin.