Bcl 2

The Plasmid Extraction Kit (D6950-02, Omega Bio-Tek, Norcross, GA, USA), RNAi-Mate Transfection Reagent (Gima Genetics, China, G04001), phosphate buffer saline (PBS) (Solarbio, China, P1010), sodium pentobarbital (P3761, Sigma Merck, Germany), eosin (E8090, Solarbio, China), hematoxylin (G1004, Servicebio, China), neutral resin (G8590, Solarbio, China), nisin staining solution (G1036, Servicebio, China), TRIzol (15596026, Ambion, USA), SYBR FAST quantitative polymerase chain reaction Master Mix (KM4101, KAPA Biosystems, China), Oligo (dT) 18 Primer (3806, TAKARA, Japan), PrimeScript II Rtase (TAKARA, Japan, 2690A), Recombinant Rnase Inhibitor (TAKARA, Japan, 2313A), 10 mM dNTP Mix (PC2200, Solarbio, China), In Situ Cell Death Detection Kit (11684817910, ROCHE, Switzerland), DAB Concentrated Kit (DA1010-2 × 3 ml, Solarbio, China), Opti-MEM (31985-062, Gibco), Lipofectamine 2000 (11668-027, Invitrogen, USA), Dual luciferase reporter gene assay kit (RG027, Biyuntian, China), caspase-9 (PAB40626, Bioswamp, China), caspase-3 (PAB30047, Bioswamp, China), BCL2-Associated X (Bax) (PAB46088, Bioswamp, China), B-cell lymphoma-2 (Bcl-2)(PAB30041, Bioswamp, China), glyceraldehyde-3phosphate dehydrogenase (GAPDH) (PAB36269, Bioswamp, China), and Goat anti-Rabbit IgG (SAB43714, Bioswamp, China) were employed in this study. A flowchart of this study is shown in Figure 1.

Proteins were extracted from the hippocampal tissue and BV2 cells. Lysis buffer and kits were used for quantification (Solarbio, Beijing, China). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) (Millipore, US). Blocking was performed with 5% skimmed milk, after which the PVDF was incubated for 12 h with primary antibodies against Bax (Bioswamp, Wuhan, China), Bcl-2 (Bioswamp), CREB (Bioswamp), p-CREB (Bioswamp), BDNF (Bioswamp), ERK1/2 (Bioswamp), p-ERK1/2 (CST), p38MAPK (Bioswamp), p-p38MAPK (Abcam), TLR4 (Bioswamp), NF-κB p65 (Bioswamp), p-NF-κB p65 (Abcam), and GAPDH (Bioswamp). The membranes were incubated with a secondary antibody (Bioswamp) for 2 h, and the protein bands were scanned.