Anti hrp rabbit

Adult brains were dissected and fixed with 4% formaldehyde in phosphate-buffered saline for 20 min whereas adult NMJ were fixed 10 min; in both cases, samples were washed 3 × 15 min with PBS+0.4% triton, blocked for 1 h with PBS+0.4% triton+ BSA 5%, incubated overnight with primary antibodies, washed 3 × 15 min, incubated with secondary antibodies for 2 h, and mounted in Vectashield mounting medium, with DAPI in the case of the brains. The primary antibodies used were anti-repo mouse (1/200; DSHB) to recognize glial nuclei, anti-bruchpilot-NC82-mouse (1/50; DSHB) to recognize the presynaptic protein bruchpilot, anti-HRP rabbit (1/400; Cell Signalling) to recognize membranes, anti-GFP rabbit (1:500; DSHB) and anti-Elav (1:100; DSHB) to recognize neuron nuclei. The secondary antibodies used were Alexa 488 or 647 (1/500; Life Technologies). Images were taken by a Leica SP5 confocal microscopy.

All tissues were treated in simultaneously for each experiment. Adult brains were dissected and fixed with 4% formaldehyde in phosphate-buffered saline for 20 min, whereas adult NMJ were fixed for 10 min; in both cases, samples were washed 3 × 15 min with PBS + 0.4% triton, blocked for 1 h with PBS + 0.4% triton + BSA 5%, incubated overnight with primary antibodies, washed 3 × 15 min, incubated with secondary antibodies for 2 h and mounted in Vectashield mounting medium with DAPI in the case of the brains. The primary antibodies used were anti-Repo mouse (1/200; DSHB, Iowa City, IA, USA) to recognize glial nuclei, anti-Bruchpilot-nc82-mouse (1/50; DSHB, Iowa City, IA, USA) to recognize the presynaptic protein Bruchpilot, anti-HRP rabbit (1/400; Cell Signaling, Danvers, MA, USA) to recognize neuronal membranes, anti-GFP rabbit (1:500; DSHB, Iowa City, IA, USA) and anti-Myc guinea pig (1/100; DSHB, Iowa City, IA, USA) to recognize the nuclear protein Myc. The secondary antibodies used were anti-mouse, -rabbit or -guinea pig Alexa 488 or 647 (1/500; Life Technologies, Carlsbad, CA, USA). Images were taken by a Leica SP5 confocal microscopy applying same conditions for each experiment.

Antibody for staining PI4P was purchased from Echelon Biosciences. Antibody against PV 3D was produced in Cameron lab. Mouse monoclonal anti-GAPDH antibody (10R-G109a) was purchased from Fitzgerald Industries. All secondary antibodies used for immunofluorescence were purchased from Invitrogen. Secondary antibodies for western blotting were purchased from Amersham GE Healthcare (rabbit anti-HRP) and Cell Signaling (mouse anti-HRP). Rabbit polyclonal anti-eIF2D antibody (12840-1-AP) and anti-eIF2A antibody (11233-1-AP) were purchased from Proteintech. Rabbit anti-phospho (S51)-eIF2α (9721) and rabbit anti-eIF2α (9722) were purchased from Cell Signaling Technology. Rabbit anti-phospho (T446)-PKR (32036) and rabbit anti-PKR (32506) were purchased from Abcam.