Bca kit

The stocked supernatants of hippocampal tissues of three rats in each group were used for Western blotting. Cells were washed by PBS and lyzed (Beyotime, Shanghai, China), followed by oscillating for 30 s and incubating on ice for 30 min. After determination of concentrations using BCA kits (Solarbio, Beijing, China), the proteins were separated on SDS-PAGE and transferred onto polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, United Kingdom). The membranes were incubated at 4°C with primary antibodies, including caspase-3 (1:1,000, Cell Signaling Technology), Bcl-2 (1:1,000, Santa Cruz Biotechnology), Bax (1:1,000, Santa Cruz Biotechnology), and β-actin (1:10,000, Cell Signaling Technology), and then probed by horseradish peroxidase-conjugated second antibodies (Zhongshan Golden Bridge, Beijing, China). The immunoreactivity was visualized using Enhanced Chemiluminescence (ECL) reagents (Amersham) and images acquired on AI-600 System (GE, United States).

Walnuts were purchased from the farmers’ market (Shihezi, China). 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH), 2,4-dinitrophenylhydrazine (DNPH), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and 8-anilino-1-naphthalenesulfonic acid (ANS) were obtained from Yuanye (Shanghai, China). Pepsin (3000 units/mg) and trypsin (250 units/mg) were purchased from Boao (Shanghai, China). The Tris-Tricine-SDS-PAGE and BCA kits were provided by Solarbio (Beijing, China). All the other chemicals were of analytical reagent grade and obtained in China.

The recombinant expression plasmids (pGEX-4T-1-VP56-1, pGEX-4T-1-VP56-2, pGEX-4T-1-VP56-3, pGEX-4T-1-VP56) were transformed into E. coli (DE3plys) and incubated in LB medium at 37°C for 4 h. Then IPTG (1 mmol/L) was added to induce expression at 37°C for 4 h.
The bacteria were collected by centrifugation (25°C, 5000 rpm, 10 min). The bacteria was resuspended with PBS (pH = 7.4) and pulverized with a high-pressure pulverizer (880 MPa). The supernatant and precipitate were collected by centrifugation (4°C, 12000 rpm, 1 h). Purification of recombinant protein (VP56-1, VP56-2, VP56-3, VP56) according to the previous method (38 (link)). Briefly, the proteins were bound to 10 mL glutathione beads (Smart-Lifesciences, China), eluted through a gradient of buffer B (50 mM Phosphate Buffer, 10 mM L-Glutathione reduced (pH = 8.0, 25°C)) and dialysis in the 50 mM Phosphate Buffer (pH = 8.0, 25°C). Validation of purified recombinant proteins using SDS-PAGE (Solarbio) and quantification of recombinant proteins using BCA kits (Solarbio).

The hippocampal tissues were homogenated using RIPA Buffer (P0013D, Beyotime, China). After centrifugation, the supernatants were collected and the protein concentrations were detected with the BCA kits (pc0020, Solarbio, China). With these steps carried out, the protein was electrophoresed and loaded onto PVDF membranes (10600023, GE Healthcare Life, USA). After sealing, the blocked membranes were reacted with primary antibodies (4°C, all night). Then, the membranes were intervened with an anti‐rabbit secondary antibody (31466, Invitrogen, USA) for another 1 h. Finally, the membranes were visualized with ECL reagents (SQ202, Epizyme, China) and analyzed with ImageJ. The primary antibodies used were phospho‐PI3K p85 alpha (Tyr607, 1:1000, AF3241), PI3K p85 alpha (1:1000, AF6241), phospho‐AKT2 (Ser474, 1:2000, AF3264), AKT2 (1:2000, AF6264), IL‐1β (1:1000, AF5103), IL‐6 (1:1000, DF6087), TNF‐α (1:1000, AF7014), and β‐actin (1:10000, AF7018) were acquired from Affinity (USA).

Proteins were isolated using RIPA buffer (KaiGene, Nanjing, Jiangsu, China) and qualified by BCA kits (Solarbio, Haidian, Beijing, China). The proteins were then separated by dodecyl sulfate, sodium salt/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Target proteins were incubated with primary antibodies: Anti-Vimentin (#10366-1-AP, CST, Danvers, MA, U.S.A.), Anti-E-Cadherin (#sc-71008, CST, Danvers, MA, U.S.A.), Anti-N-Cadherin (#ab18203, Abcam, Pudong, Shanghai, China), Anti-Caspase-9 (#ab325539, Abcam, Pudong, Shanghai, China), Anti-Caspase-3 (#9662, CST, Danvers, MA, U.S.A.), Anti-β-catenin (#51067-2-AP, CST, Danvers, MA, U.S.A.), Anti-Cyclin D1 (#2922, Abcam, Pudong, Shanghai, China), Anti-c-Myc (#ab39688, Abcam, Pudong, Shanghai, China), Anti-GAPDH (#60004-1-Ig, CST, Danvers, MA, U.S.A.). Afterward, matched secondary antibodies was used to incubate the membranes, followed by examination using ECL analyses kits (KeenBio, Xuhui, Shanghai, China).

We first separated proteins from tissues or cells using RIPA lysis buffer (Merck Millipore, Waltham, MA, USA) and then assayed the corresponding protein concentrations employing BCA kits (Solarbio Co.). After electrophoresis of the samples, we transferred them to polyvinylidene difluoride (PVDF) membranes supplied by Merck Millipore. These membranes were then incubated for 2 h in a solution containing 5% skim milk. After adding the primary antibody solution to the samples, they were incubated overnight at 4°C. After a 2-h incubation with the secondary antibody, the membranes were washed three times with TBST. The bands were then observed. We used the imaging system from Bio-Rad Laboratories (Hercules, CA, USA). The following antibodies were used: anti-PYGB (1:1000, Proteintech, #12075), anti-MEK1/2 (1:5000, Proteintech, #11049), anti-ERK1/2 (1:2000, Proteintech, #11257), anti-P-ERK1/2 (1:1000, Proteintech, #28733), anti-P-MEK1/2 (1:1000, Cell Signaling Technology, #9154), anti-α-Tubulin (1:2000, Proteintech, #66031), HRP-goat anti-rabbit IgG (1:2000, Proteintech, #SA00001-2), and HRP-goat anti-mouse IgG (1:2000, Proteintech, #SA00001-1).

First of all, the WB experiment involved in this paper adopted the trimmed membrane for western blotting, which is a routine experimental method in our laboratory. Second, blots were cut off before hybridization with antibodies in the paper involved protein stripe images, we have provided the original blots. The specific method is the proteins were quantified with BCA kits (Solarbio, PC0020), separated by polyacrylamide gel electrophoresis (Bio-Rad), and transferred to PVDF membrane (Millipore, USA) after cleavage of the rat prefrontal cortex with a mixture of protease inhibitors (RIPA:PMSF = 100:1). Then, 5% skimmed milk powder was used for sealing after electroporation, followed by incubation with antibodies against NLRP3 (Novus, NBP2-12446, CO, USA), Caspase-1 (Novus, NB100-56564, Cambridge, UK), IL-1β (BIOSS, bs-20449R, Beijing, China), IL-18 (Proteintech, 10663-1-AP, Wuhan, China), IL-6 (BIOSS, bs-0782R), TNF-α (Abcam, ab178846, Cambridge, UK), β-actin (Proteintech, 20,536–1-APa), GAPDH (Proteintech, 10494-1-AP), LC3B (HuaBio, ET1701-65, Hangzhou, China), and SQSTM1/p62 (HuaBio, R1309-8).