Antiphosphorylated ire1

Eyes were oriented, fixed in Davidson's fixative (8% Formaldehyde, 31.5% Ethanol, 2 M Acetic Acid), embedded in paraffin and sectioned. Apoptotic nuclei were detected by TdTmediated dUTP terminal nick-end labelling kit (TUNEL, fluorescein; Roche) according to the producer's protocols. Primary antibodies were used as follows: anti-AIF (1:100; Sigma), antiphosphorylated-IRE1 (1:200; Novus Biologicals), anti-phosphorylated-PERK (1:100; Cell Signaling). Secondary antibodies were Alexa Fluor ® 568 anti-mouse and anti-rabbit antibodies (1:1000; Molecular Probes). Nuclei were stained with 4′,6-diamidino-2phenylindole (DAPI). Slides were mounted with mowiol 4-88 (Sigma) and analyzed at a Zeiss Axio Imager A2 microscope. Quantification of dying cells was performed by counting all TUNEL labeled cells in the photoreceptor cell layer in at least three entire retinal sections, with a dorsal-ventral orientation, passing through the optic nerve derived from different animals. The number of photoreceptors double labelled with anti-AIF and TUNEL was counted by staining of nuclei with DAPI and was used to calculate percentages of dying cells, as previously published [23] .

Retinas were lysed as previously described to obtain nuclear-enriched proteins and total cellular proteins [24] . The purity of enriched lysates was checked by immunoblotting using a nuclear marker (anti-Histone H3 1:3000; Bethyl Laboratories) or a cytosol marker (antipan-actin, 1:3000; Millipore). Equivalent amounts of protein extracts (20 g) were resolved using SDS-PAGE and immunoblottings were performed following standard procedures. The antibodies used for western blotting were: anti-II-spectrin (AA6, 1:2000; Enzo Life) [25] , anti-AIF (1: 1000; Calbiochem) [26] , anti-Caspase7 (1:1000; Cell Signaling), anti-eIF2 (1:1000; Cell Signaling), anti-NRF2 (1:2000; Invitrogen), anti-phosphorylated-IRE1 (1:2000; Novus Biologicals), anti-phosphorylated-PERK (1:1000; Cell Signaling), antiphosphorylated-eIF2 (1:1000; Cell Signaling). Antibodies specificity is shown in Fig. S2.
Each blot analyzed proteins derived from 4 retinas pooled together and 3 independent pools from 3 different litters were used as biological replicates, one representative blot is shown.
The entire blots are shown in Fig. S3.