Rat anti eomes

Chicken anti-GFP (1:2000; Abcam, #AB13970); Goat anti-TOM (1:300; Sicgen, #AB8181-200); Rabbit anti-RFP (1:100; Abcam, #AB62341); Mouse anti-PAX6 (1:300; Thermoscientific, #MA1-109); Rat anti-EOMES (1:500; Invitrogen, #14-4875-82); Rat anti-EOMES (1:300; eBioscience, #144875-82); Rabbit anti-KI67 (1:250; Abcam, #AB15580); Rabbit anti-NEUROD2 (1:1000; Abcam, #AB104430); Mouse anti-RORB (1:200; Perseus Proteomics, #PP-N7927-00); Rabbit anti-CUX1 (1:250; Santa Cruz, #sc13024), Rabbit anti-CASPASE3 (1:3000; Cell signaling technology, #9662S), Mouse anti-BRN2 (1:200; Santa Cruz, #sc393324). All secondary antibodies were 488/555/647 conjugated (1:500, Invitrogen).

Cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, subjected to SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline with Tween 20, incubated in primary antibodies overnight including rabbit anti-Eomes CTD (Abcam, ab23345, 1:2,000), rabbit anti-Eomes NTD (Santa Cruz, sc-98555, 1:1,000) and rat anti-Eomes (eBioscience, 14–4,876, 1:1,000). Secondary antibodies were donkey anti-rabbit horseradish peroxidase (GE Healthcare NA934, 1:2,000) and goat anti-rat horseradish peroxidase (GE Healthcare NA935, 1:2,000). Blots were developed by chemiluminescence using Amersham ECL Prime Detection Reagent (GE Healthcare).