P perk

The hippocampus was homogenized in a lysis buffer containing a protease inhibitor cocktail (10 μL/ml, GE Healthcare Biosciences, PA, United States). The complex was centrifuged at 12,000 rpm/min at 4°C, and supernatants were removed to determine protein concentrations, calculated according to the standard curve. Proteins were separated on 10 or 12% SDS-polyacrylamide gels at 80 V for 2 h. After separation at 300 mA for 1.5 h, the protein bands were transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for 1.5 h. After washing three times with Tris-buffered saline with 0.05% Tween 20, the membranes were incubated with the primary antibodies overnight: JNK1/2, p-JNK1/2, GAPDH, superoxide dismutase 2 (SOD2), IL-6 (Proteintech, Chicago, IL, United States), Bax, Bcl-2, C-caspase3, postsynaptic dense protein 95 (PSD95), Nrf2, p-CREB133, BDNF, IL-1β, synapsin-1 synaptophysin, GRP78, ATF, protein kinase R-like endoplasmic reticulum kinase (PERK), p-PERK, p-IRE1α, CREB, p-CREB129, PKA, and IL-10 (Abcam, Cambridge, United Kingdom). The following day, membranes were incubated in corresponding secondary antibodies for 1 h at room temperature. Signals were visualized using the ChemiDocXRS + Imaging System (Bio-Rad). Blot bands were quantified using ImageJ software densitometry and expressed as relative density to GAPDH.

The primary antibodies sarco/endoplasmic reticulum calcium ATPase (SERCA, 1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA, #271669), C/-EBP homologous protein (CHOP, 1:100, Santa Cruz Biotechnology, #7351), Bcl-2 (1:500, Cell Signaling Technology, Danvers, MA, USA, # 4223S), p-PERK (1:500, Abcam, Cambridge, UK, #156919), cytochrome c (1:100, Abcam, #90529), and β-actin (1:2000, Santa Cruz Biotechnology, #47778) were purchased and maintained overnight at 4 °C. Same quantities of protein were divided on 8–10% SDS (sodium dodecyl sulfate-polyacrylamide) gels and electro-transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). Process of blocking was performed with 10% non–fat milk in TBS–T for 1 h at RT (room temperature). The membranes were then washed 3 times with TBS-T and probed with the corresponding secondary antibodies conjugated to HRP (horse radish peroxidase, Santa Cruz, CA, USA) at RT for 1 h. After washing, the blots were developed with ECL reagents (Pierce) and exposed using Kodak X–OMAT AR Film (Eastman Kodak, Rochester, NY, USA) for 1–5 min.

TMF has been isolated from the leaves of M. exotica and purified by repeated column chromatography and recrystallization in acetoacetate. The purity was found to be more than 98%. Fetal bovine serum (FBS), Dulbecco's modified Eagle's minimum essential medium (DMEM) (low glucose), penicillin, and streptomycin were purchased from Gibco (Life Technologies, NY, USA). GRP78, PERK, p-PERK, eIF2α, p-eIF2α, ATF6, IRE1α, XBP1s, CHOP, JNK, GSK-3β, Bax, Bak, and GAPDH monoclonal antibodies and the peroxidase-conjugated secondary antibody were obtained from Abcam (Cambridge, UK).

For the Western blot analysis, protein extraction was performed using RIPA protein lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor cocktail and PMSF. The extracted protein was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% skim milk for 1 hour at room temperature. Subsequently, the primary antibody was used for incubation overnight. Prior to detection, membranes were washed with PBST and incubated with secondary antibodies in blocking buffer for 2 hours. The antibodies used for western blots were as follows: PCK2 ( Abclonal, A8446), IREa (CST, 3294S), P-IREa (Novus, NB100-2323), PERK (CST, 3192S), P-PERK (Abcam, ab192591), ATF6 (CST, 65880S), and GAPDH ( Proteintech, 60004-1-Ig).

Primary antibodies for SERCA (1:200, #271669; Santa Cruz Biotechnology, CA, USA), C/-EBP homologous protein (CHOP, 1:100, Santa Cruz Biotechnology, #7351), Bcl-2 (1:500, # 4223S; Cell Signaling Technology, Danvers, MA, USA), p-PERK (1:500, #156919; Abcam, Cambridge, U.K.), caspase-3 (1:200, #56053; Santa Cruz Biotechnology), and β-actin (1:2000, #47778; Santa Cruz Biotechnology) were purchased and maintained overnight at 4 °C. The detailed protocol can be found in our previous study [27 (link)].

The procedures for protein extraction, protein concentration determination, conventional SDS-PAGE, protein band visualization, and protein band intensity measurement were performed according to our reported methods [20 ,21 (link)]. The used antibodies which were recognized included Bcl-2 (1:1000), Mcl-1 (1:1000), Noxa (1:1000), Bax (1:1000), PERK (1:1000), eIF2α (1:1000), LC3 (1:1000), p62 (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), P-PERK (1:500, Threonine-982), P-YAP1 (1:500, Serine-127) (Abcam, Cambridge, UK), P-eIF2α (1:500, Serine-51), YAP1 (1:1000) (Cell Signaling, Danvers, MA, USA), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH, 1:3000) (R&D Systems, Minneapolis, MN, USA).