Striatal Protein Extraction and Western Blot

After pharmacological manipulation, the striatum (including the nucleus accumbens and dorsal striatum) of one hemisphere was extracted as previously described (Puighermanal et al., 2016a (link)), sonicated in 300 μl of 10% sodium dodecyl sulfate (SDS), and boiled at 100°C for 10 min. Protein quantification and western blots were performed as described (Biever et al., 2015 (link)). Primary antibodies against p-eIF2α (Ser51) (1:1000; Cell Signaling, #3398), eIF2α (1:1000; Cell Signaling, #5324), p-eEF2 (Thr56) (1:1000; Cell Signaling, #2331), p-p70S6K (Thr389) (1:1000; Cell Signaling, #9234), p-4EBP1 (Thr37/46) (1:500; Cell Signaling, #2855), 4EBP1 (1:500; Cell Signaling, #9644), OPHN1 (1:1000; Cell Signaling, #11939), ATF4 (1:1000; NeuroMab, #75-345), MAP2 (1:2000; Sigma, #M4403) from Sigma, CaMKIIa (1:1000; Millipore, #05-532), puromycin [1:1000; (David et al., 2012 (link))], and β-actin (1:40000; Abcam, #AB6276) were used. The optical density of the relevant immunoreactive bands (or for all the bands for puromycin staining) was quantified after acquisition on a ChemiDoc XRS System (Bio-Rad) controlled by Image Lab software version 3.0 (Bio-Rad).