P eif2α

The human chondrocyte cell line C28/I2 was used [34] (link). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Lonza) containing 10% FBS (Hyclone) and 1% penicillin/streptomycin (Lonza), and maintained at 37°C and 5% CO₂ in a humidified incubator. Neon transfection system (Invitrogen) was used to transfect Src biosensors into the cells. After transfection, the cells were transferred to a type I collagen-coated glass bottom dish or µ-slide cell culture chamber (Ibidi) and incubated in DMEM containing 0.5% FBS for 24–36 h before imaging experiments. For Western blotting, cells were grown in the presence and absence of salubrinal or guanabenz and lysed in a radioimmunoprecipitation assay (RIPA) buffer. Isolated proteins were fractionated using 10% SDS gels and electro-transferred to Immobilon-P membranes. The membrane was incubated for 1 h with primary antibodies followed by 45 min incubation with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling). We used antibodies against eIF2α (Cell Signaling), phosphorylated eIF2α (p-eIF2α; Thermo), and β-actin (Sigma). Signal intensities were quantified with a luminescent image analyzer (LAS-3000, Fujifilm).

Protein lysates were processed for immunoblots as described (Rutkowski et al, 2006 (link)). Primary antibodies include: CHOP (Santa Cruz sc-7351 RRID:AB_627411 for flow cytometry or Proteintech 15204-1-AP RRID:AB_2292610 for western blot), PARP (Cell Signaling Technology 9542 RRID:AB_2160739), ATF4 (Cell Signaling Technology 11815 RRID:AB_2616025), PeIF2α (Thermo 44-728G), total eIF2α (Cell Signaling Technology 9722 RRID:AB_2230924), and Calnexin (loading control; Enzo ADI-SPA-865 RRID:AB_10618434). Nanoluciferase activity was assessed using the Nano-Glo In-Gel Detection kit (Promega, USA) following the manufacturer’s protocol. qRT-PCR, including primer validation by standard curve and melt curve analysis, was also as described (Rutkowski et al, 2006 (link)). Briefly, RNA was isolated using Trizol (Thermofisher, USA) following the manufacturer’s protocol, and RNA concentrations were evaluated using the Qubit RNA Broad Range kit (Invitrogen, USA). 400 ng of RNA was used for reverse transcription using PrimeScript RT Master Mix (Takara, USA). PCR reactions were performed using TB Green Premix Ex Taq (Takara, USA). Gene expression was normalized against the average of two loading controls (Btf3 and Ppia).

We extracted hypothalamic tissue protein and the Western blot test according to the previously described method [37 (link)]. In this part of the experiment, we used the following antibody concentrations: β-actin, 1:8000 (Proteintech, 66009-1-Ig); β-tubulin, 1:5000 (Abmart, M20005); PER2, 1:3000 (Boster, PB0347); ERK, 1:8000 (Sigma, M5670); p-ERK, 1:8000 (Sigma, M8159); NRF2, 1:1000 (Boster, PB9290); p-EIF4E, 1:2000 (Novus Biologicals, NBP266802); p-EIF2α, 1:1000 (Thermo Fisher Scientific, MA5-15133).

Western blots were performed to detect protein levels in the ipsilateral cerebral cortex and primary microglia cells. The ipsilateral cerebral cortex tissues were homogenized and lysed with RIPA buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Abcam) and treated cells were lysed using a Mammalian Cell Lysis kit (Sigma). The extracted proteins were then separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide and electrically transferred to PVDF membranes (Millipore). The membranes were then blocked with TBST with 5% non-fat dry milk for 1 h at room temperature. The western blots were probed with primary antibodies recognizing the following proteins and further incubated with corresponding secondary antibody (1:10000; Invitrogen): PTP1B (1:1000; Abcam); p-PERK (Thr982, 1:1000; Invitrogen); PERK (1:1000; Invitrogen); p-eIF2α (Ser51, 1:1000; Invitrogen); eIF2α (1:1000; Invitrogen); p-IRE1 (Ser724, 1:1000; Abcam); IRE1 (1:2000; Abcam); ATF6 (1:2000; Abcam); LC3-I/II (1:1000; Cell Signaling); beclin-1 (1:1000; Cell Signaling); β-actin (1:5000; Abcam); and GAPDH (1:5000; Abcam). The levels of protein expression were analyzed using Image J software (version 1.49) normalized to β-actin and GAPDH. Phosphorylated protein levels were evaluated compared to total protein levels.

Transfected cells were grown in 6-well plates and harvested for protein extraction. Cells were lysed using the CelLytic M reagent (Sigma-Aldrich, St. Louis, MO, USA) and scraped from the plates. Lysates were separated in 12% NuPAGE Bis-Tris gels (Invitrogen) and transferred to the PVDF membrane using Trans-Blot SD semi-dry transfer cell (BioRad, Hercules, CA, USA). Membranes were blocked for 2 h using 5% dry milk in TBST (0.02 M Tris-HCl, 0.9% NaCl, 0.05% Tween 20, pH 7.6). Polyclonal antibody against phosphorylated eIF2α (p-eIF2α) (Invitrogen), actin (Sigma) and mouse anti-c-myc monoclonal antibody was diluted in 2.5% dry milk in TBST and incubated overnight at 4 °C. Horseradish peroxidase (HRP) conjugated anti-rabbit or anti-mouse antibody (Cell Signaling, Danvers, MA, USA) diluted 1:2000 were added and incubated for 1 h. Final detection was achieved using the ECL Plus™ Western Blotting (WB) detection reagents and a Typhoon scanner (Amersham Biosciences, Little Chalfont, UK).
Quantification of eIF2α phosphorylation after transfection of pcDNA-wtPKR and pcDNA-mutPKR in EPC (2 experiments) and AGK cells (1 experiment) was done at 16, 24, and 40 h post transfection. The amount of p-eIF2α measured by densitometry (Typhoon Imager, GE Healthcare, Chicago, IL, USA) was quantified with ImageJ software, and the value was normalized against β-actin levels.

Primary hippocampal cultures were prepared as described above. After 18–19 DIV, the cells were incubated with 20 µM BIC, 1 µM TTX or no drug (Ctrl) for 1, 3 or 7 days as indicated, subsequently washed with ice-cold PBS (Invitrogen) and scraped in PBS supplemented with 0.5% SDS, 0.5% Triton-X100, protease inhibitor cocktail (Roche), Halt phosphatase inhibitor (Thermo Scientific) and benzonase (2 µL/mL, ≥250 units /µL, Sigma). The lysates were cleared by centrifugation at 17,000 x g for 15 min. Protein concentration was determined by BCA assay (Thermo Scientific). Equal protein amounts were used for western blot analysis. Proteins were separated by electrophoresis and immunoblotted with antibodies against Bdnf (1:400, Santa Cruz Biotechnology, Ref: sc-546), Homer1 (1:1000, Synaptic Systems, Ref: 160003), GluA1 (1:1000, Synaptic Systems, Ref: 182003), Synaptopodin (1:1000, Synaptic Systems, Ref:163002), Camk1g (1:2000, Abcam, Ref: ab227209), β-actin (1:3000, Sigma, Ref: A5316), eIF2α (1:5000, Cell Signaling Technology, Ref: L57A5) and p-eIF2α (1:2500, Invitrogen Ref: 44728G). For western blots against eIF2α and p-eIF2α, phosphatase inhibitor was added to all solutions. Three biological replicates were performed.