P eif2α ser51

An RNeasy Plus kit (Qiagen, Germantown, MD, USA), a high capacity cDNA reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA), and a Power SYBR Green PCR master mix kit (Applied Biosystems) were employed with listed PCR primers (Table 1). For detecting GFP-labeled 4T1.2 cells in the right femur, total DNA was isolated with QIAamp DNA mini kit (Qiagen) for qPCR.
For Western blotting, cells were lysed by a radio-immunoprecipitation assay buffer (Santa Cruz). Proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Antibodies against ATF4, caspase 3, cathepsin K, eIF2α, p-eIF2α (Ser51), LC3A/B II, NFATc1, Chk1, p-Chk1 (Ser296) (Cell Signaling, Danvers, MA, USA), TRAP (Abcam, Cambridge, MA, USA), and β-actin (Sigma) were utilized.

EIF2α, p-EIF2α (Ser51), RelA, NFATc1, c-fos, ATF3, HSF1, p-HSF1 (Ser320), p24, Lamin A/C, β-actin antibodies and secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Acetylated lysine antibody, p300, CDK9 and Cyclin T1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human FITC conjugated anti-CD25 and PE conjugated anti-CD69 antibodies were purchased from BD Biosciences (San Jose, CA, USA). PHA, poly I:C, STA-4783, thapsigargin, prostratin, PMA, ionomycin, SAHA, JQ1, dilazep, TNF-α, hemin, salubrinal, MG-132, BAY 11-7082, CsA, C646 and EX527 were from Sigma-Aldrich (St. Louis, MO, USA). Resveratrol, parthenolide (PTN), Ver-155008, KRIBB11, 17-DMAG and NVP-AUY922 were from Merck Calbiochem (Darmstadt, Germany). PEZ-HSF1 was purchased from ViGene Bioscience Inc (MD, USA). NL4-3E-R-luc plasmid, J-Lat 10.613 (link), U141 (link) and ACH242 (link) cell lines were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program.
J-Lat 10.6, U1 and ACH2 cell lines were maintained in RPMI1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (Gibco) at 37 °C with 5% CO₂. High temperature treatment (39.5 °C) was operated by putting cells in another incubator which temperature was adjusted to 39.5 °C.

eIF2α, P-eIF2α (Ser51), Total S6K1, P-S6K1 (T389), Cleaved PARP, total and cleaved caspase-3 antibodies were purchased from Cell Signaling Technologies, ATF4 (SC-200) from Santa Cruz and MAP1LC3B antibody from Novus. Antibody Puromycin (clone 12D10) was kindly given by Phillipe Pierre (Centre d'Immunologie de Marseille-Luminy, France).

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).

Antibodies for IRE1α (#3294), PDI (#3501), PERK (#3192), eIF2α (#5324) and its phosphorylated form at ser51 (#3398, p-eIF2α^ser51) GRP78 (used in the cancer experiment, #3177), P62 (#5114), Bcl-2 (#3498), p-ULK1^ser757 (#14202), ULK1 (#8054), p-JNK^{Thr183/Tyr185} (#4668), JNK (#9252), ATF4 (#11815), Beclin-1 (#3495), caspase 12 (#2202), and cleaved caspase 3 (#9661) were purchased from Cell Signaling Technology. GAPDH (ab9485) antibody was purchased from Abcam (Cambridge, United Kingdom). LC3I and LC3II were measured by antibody (L7543) that was purchased from Sigma-Aldrich (St. Louis, MO, United States). Monoclonal primary antibodies were used for the detection of heat shock protein 70 (HSP70, StressGen, SPA-810), heat shock protein 60 (HSP60, StressGen, SPA-806), heat shock protein 90 (HSP90, StressGen, SPA-835), heat shock protein 47 (HSP47, StressGen, SPA-470) and thioredoxin interacting protein (TXNIP and MBL). Polyclonal primary antibodies were used to detect thioredoxin (TRX, IMCO, and ATRX-06), actin (Sigma, A-2066), heat shock protein 25 (HSP25, StressGen, and SPA-801), glucose-regulated protein 78 (GRP78, StressGen, SPA-826, used in the acute experiments). Horseradish peroxidase conjugated IgG secondary antibodies were used (Jackson ImmunoResearch Laboratories, PA, United States and StressGen and Zymed, San Francisco, CA, United States).