Ready gel precast gel

2-DGE was performed on Bio-Rad PROTEAN IEF cell. The ReadyPrep 2-D Starter Kit, ReadyStrip IPG Strips, Ready Gel precast gel, mineral oil, 10X Tris/glycine/SDS Buffer, and paper wicks were purchased from Bio-Rad (USA). Buffers were prepared according to the kit protocol. 125 µL of freshly prepared rehydration/sample buffer for 7 cm IPG strip was added in a conical centrifuge tube containing the sample pellet, and vortexed to dissolve the pellet. The sample was centrifuged to settle down the fine particles. The whole procedure was performed according to the kit protocol. Gel images were taken through Gel DOC 800 system (Bio-Rad, USA).

Samples from cell lines were scraped on ice with RIPA cell lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Roche). Proteins were separated by a Ready Gel precast gel (Bio-Rad) and transferred onto a nitrocellulose membrane. Blocking was done with 5% milk, 1 h at RT. Primary antibody incubations were performed overnight at 4° for both polyclonal NEUROD1 (1:500, PA5-47381; Invitrogen) and monoclonal β-tubulin (1:20,000, SAP.4G5). Secondary antibodies (polyclonal rabbit anti-mouse immunoglobulins/HRP [1:1,000, P0260; Dako] and polyclonal rabbit anti-goat immunoglobulins/HRP [1:2,000, P0160; Dako]) were incubated for 1 h at RT. All antibody dilutions were made to 5% milk. Proteins were detected by enhanced chemiluminescence (Bio-Rad).