Anti phospho eif2α ser51

For Western immunoblotting analysis, infected neurons were collected in 1× LDS loading buffer (Invitrogen) containing 2.5% β-mercaptoethanol and boiled for 10 min. Proteins were separated by electrophoresis in 10% NuPAGE gels and immunoblotting was carried out as previously described (Liu et al., 2014 (link)). Densitometric analysis of the bands was performed using the ImageJ program. Comparison of data and calculation of p values were performed by using Student’s t-test.
The following primary antibodies were used: rabbit anti-ATF4 (1:1000, Cell Signaling), anti-phospho-eIF2α (Ser51; 1:1000, Cell Signaling) and mouse anti-GAPDH (1:2000; Imgenex). The anti-ATF4 has been validated by loss of signal after ATF4 knockdown with shRNAs (Liu et al., 2014 (link); Pasini et al., 2015 (link)).

Muscle lysates were prepared in RIPA buffer (50-mM Tris-HCl, 150-mM NaCl, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) containing protease inhibitor cocktail (Nacalai Tesque) and phosphatase inhibitor cocktail (Biotool) using a Polytron homogenizer (Kinematica). The protein concentrations of lysates were measured using the bicinchoninic acid method, and immunoblot analyses were performed as described previously [12 (link)]. Proteins were detected using anti-PERK, anti-phospho-PERK (Thr980), anti-eIF2α, anti-phospho-eIF2α (Ser51), anti-Akt, anti-phospho-Akt (Ser473), anti-p70 S6 kinase (S6K), anti-phospho-S6K (Thr389), anti-eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4EBP) 1, and anti-phospho-4EBP1 (Thr37/46), which were purchased from Cell Signaling Technology. GAPDH, which was purchased from MBL international, was used as a loading control. Bands were detected using WesternSure ECL Substrate (Li-Cor Biosciences), and images were acquired using an EZ-Capture II Cooled CCD Camera System (ATTO Corp.). The band intensity was determined using ImageJ software.

Crude protein extracts (50 μg) were resolved by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gels and then transferred to PVDF Immobilon-P membranes (Bio-Rad, Mississauga, ON). Blots were blocked using 5% milk powder (Selection brand, Marché Jean-Talon, Montréal, QC) and incubated with antibodies using a dilution of 1:500 for anti-β-actin, a 1:250 dilution for anti-CaMKII beta subunit and anti-CaMKII gamma subunit (Santa Cruz Biotechnology, Santa Cruz, CA), a 1:500 dilution for anti-CHOP, a 1:1000 dilution for anti-LC3B, anti-ATF4, anti-phospho eIF2α (Ser51), and a 1:10000 dilution for anti-eIF2α (Cell Signaling). Immunoreactive proteins were exposed to anti-rabbit or anti-mouse horseradish peroxidise-conjugated secondary antibodies (Millipore) that were diluted 1:5000. Antigen-antibody complexes were detected using Immobilon ECL Western Chemiluminescent HRP Substrate (Millipore) and recorded with a VersaDoc imaging system (Bio-Rad).

Cells were lysed in 50 mM Tris/HCl pH 7.6, 150 mM NaCl containing 1% Triton-X100, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and clarified by centrifugation at 15,000 g for 15 min. Proteins were resolved by SDS PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). For Western blot analysis, membranes were blocked in PBS, 0.1% Tween 20, 5% BSA for 1 h followed by incubation overnight at 4°C with one of the following rabbit polyclonal antibodies: anti-eIF2α (cat #9722) or anti-phospho-eIF2α (Ser51, cat #9721) (Cell Signaling). After being washed three times in PBS, 0.1% Tween 20, membranes were incubated for 1 h with goat anti-rabbit horseradish peroxidase-conjugated IgG (Sigma) in blocking solution. Membranes were then washed extensively in PBS, 0.1% Tween 20 and developed by using enhanced chemiluminescence detection reagents (Amersham Biosciences) in accordance with the manufacturer’s protocol.

Western immunoblotting was performed as previously described [15 (link)]. The primary antibodies used in this study were anti-cleaved caspase 3 and anti- phospho-eIF2α (Ser51) (Cell signaling tech, Danvers), anti-GAPDH (Proteintech, Chicago), anti-ATF4 (Wanlei Bio, Shenyang, China), anti-GADD153/CHOP (Wanlei Bio, Shenyang,China), anti-ATF3, anti-eIF2α, anti-pPERK and anti-PERK (Santa Cruz biotech, Santa Cruz). HRP conjunct secondary Antibodies were purchased from Jackson immune Research-laboratories (Western Grove, PA). The PVDF membrane was purchased from Millipore (Millipore, Billerica) and ECL substrate was purchased from Thermo Fisher (Thermo Fisher, Waltham).