Phosphor eif2α

Cells were lysed in lysis buffer consisting of 50 mM HEPES, 40 mM NaCl, 2 nM EDTA, 0.5% Triton X-100, 1.5 mM Na₃VO₄, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium beta-glycerophosphate and 1 tablet of protease inhibitor. Cell lysates were separated on a 4–20% Tris-gel, transferred to nitrocellulose membrane and blocked in PBST +5% milk for an hour. Membranes were incubated with primary antibody at the following concentrations: ATF4 (sc-200; Santa Cruz, Santa Cruz, CA) 1:100, phosphor-eIF2α (5324; Cell Signaling, Danvers, MA) 1:200, eIF2α (3597; Cell Signaling) 1:1000, p-PERK 1:200 (sc-32577; Santa Cruz), PERK 1:200 (sc-13073; Santa Cruz), BiP (3177; Cell Signaling) 1:1000, eIF4E (610270; BD Biosciences, San Jose, CA) 1:1000 in PBS + 5% BSA overnight at 4°C. Next day washed and incubated with 1:2000 HRP-conjugated secondary before development with SuperSignal West Pico Substrate (#34080; Pierce, Rockford, IL).

BLM and melatonin were from Sigma Chemical Co. (St. Louis, MO). XBP-1, α-SMA, GAPDH and phosphor-JNK antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA). GRP78, phosphor-IRE1α, ATF6, and phosphor-eIF2α antibodies were from Cell Signaling Technology (Beverley, MA). Chemiluminescence (ECL) detection kit was from Pierce Biotechnology (Rockford, IL). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO) if not otherwise stated.

Twenty-five-day heat-treated male fly whole bodies or heads were homogenized in 4× lithium dodecyl sulfate (LDS) loading buffer and 10× sample reducing agent (Thermo Fisher Scientific, Waltham, MA, USA) for Western blot analysis. To separate the total protein extract, 4–12% gradient SDS-PAGE (Invitrogen, Carlsbad, CA, USA) was used, which was transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The primary antibodies used were as follows: total eIF2α (1:200; Abcam, Cambridge, UK, ab26197-100), phosphor-eIF2α (1:1000; Cell Signaling, Danvers, MA, USA, 9721S), β-actin (1:5000; Cell Signaling, Danvers, MA, USA, 4967S), or HSP70 (1:1000; Enzo Life Sciences, Farmingdale, NY, USA, SPA-822). Secondary antibodies used were as follows: goat anti-rabbit IgG horseradish peroxidase (HRP) and goat anti-mouse IgG HRP conjugate (1:2000; Millipore, Burlington, MA, USA, AP307P, AP308P). Proteins were detected using an ECL-Plus kit (Thermo Fisher Scientific, Waltham, MA, USA).

The puromycin-resistant pLKO lentiviral vector containing a short hairpin RNA (shRNA) against Zbtb38 (shZbtb38-1: 5′-ATCACGAGCAGAGGCATATTC-3′; shZbtb38-2: 5′-ATACGGGAGAAAGACGATATC-3′) were used to knock down Zbtb38 gene in SH-SY5Y cells. siRNAs against ATF4 (shATF4-1: 5′-CUGCUU ACGUUGCCAUGAUTT-3′; shATF4-2: 5′-CCCUUCAG AUAAUGAUAGUTT-3′; shATF4-3: 5′-CCUGAAAGA UUUGAUAUAATT-3′) were used to deplete ATF4 gene in SH-SY5Y cells. Scramble shRNA (shNC) and siRNA (siCONT) were used as negative control for knockdown experiment. To construct the Zbtb38 lentiviral expression vector, the full-length human Zbtb38 was cloned into pLVX-Puro vector (Clontech, Mountain View, CA) according to the manufacturer's instructions. Zbtb38 and ATF6 were purchased from NovusBio (Littleton, CO) and Peprotech (Rocky Hill, NJ), respectively. ATF4, GAPDH, eIF2α, phosphor-eIF2α, and cleaved-Caspase3 antibodies were procured from Cell Signaling (Beverly, MA). In Situ Cell Death Detection Kit-POD was purchased from Roche (Basel, Switzerland).

In total, 10⁶ Cells were lysed in Cell Signalling technololgies lysis buffer (Cell Signalling) supplemented with protease and phosphatase inhibitors. DNA was sheared by passing the sample through a fine gauge needle 8 times before centrifugation for 10 min at 14,000 x g. The supernatants were retained and stored at −20 °C before further use. NuPAGE sample buffer (Invitrogen) was added to samples before separation on 4-12% polyacrylamide gels (Invitrogen, NP0321). Proteins were then transferred to PVDF membranes followed by blocking with 5% milk/Tris Buffered Saline with 0.1% tween (TBS-T), incubation with primary antibodies in 5% BSA/TBS-T overnight at 4 °C then corresponding secondary antibodies in 5% milk/TBS-T for 1 h at room temperature before incubation of the membranes with ECL (BioRad,. 1705061) and visualisation using an Amersham Imager (GE Healthcare). Further analysis was performed in ImageJ. Primary antibodies were used at the following concentrations: ATF6 (Abcam, AB122897), 1:500, total Histone 3 (Cell Signalling, 44995), 1:5000, MPI (Santa Cruz Biotech, SC393477), 1:1000, PARP (Cell Signalling, 56255), 1:500, EIF2α (Cell Signalling, 9722), 1:1000, phosphor-EIF2α (Ser51, Cell Signalling, 9721), 1:1000. Uncropped and unprocessed scans of the blots are available in Supplementary Information.