Ab131465

To collect protein from in vivo retinas, mice were deeply anesthetized and euthanized by inhalation overdose of CO₂ 2 weeks after virus injection, and after retinal dissection, proteins were extracted in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific, Waltham, MA, USA). For Western blots, the samples were boiled at 100 °C for 5 min, and equal amount of proteins from cell lysates were loaded on the SDS PAGE. Then proteins were transferred to PVDF membrane and incubated with primary antibody at 4 °C overnight. Primary antibodies used for these experiments included anti-Elk-1 (1:1000, ab131465; Abcam, Cambridge, UK, sc-365876; Santa Cruz Biotechnology), anti-Flag (1:1000, 14793; Cell Signaling Technology), anti-GAPDH antibody (1:2000; Cell Signaling Technology). Secondary antibodies were horseradish peroxidase (HRP)–conjugated anti-mouse IgG (1:2000; NA-931; GE Healthcare Life Science, Little Chalfont, United Kingdom) and anti-rabbit IgG antibody (1:3000; NA-934; GE Healthcare Life Science). Immunopositive bands were visualized with enhanced chemiluminescence (ECL; Thermo Fisher Scientific) and imaged on an LAS-3000 (Fujifilm, Tokyo, Japan). Densitometry was performed with ImageJ (http://imagej.nih.gov/ij/; NIH, Bethesda, MD, USA).

Thirty µg of protein was resolved on a polyacrylamide-SDS gel and electro-transferred to PVDF. Antibodies used in this study were, anti-ELK1 (Santa Cruz #sc-355: referred to as Ab^SC), anti-ELK1 (Abcam #ab131465: referred to as Ab^Ab), anti-HA (clone 16B12, Covance), anti-eIF2α (Invitrogen #44728G), anti-phospho (Ser51) eIF2α (Cell Signaling #9721), anti-actin (clone C4, Millipore) and goat anti-mouse or rabbit HRP secondary antibody (Bio-Rad). Blots were developed using the Super Signal Substrate (Thermo Scientific) and quantified using the Quantity One software package (Bio-Rad).