Anti p jnk and jnk

20 μg of brain homogenates, TIF (Triton-Insoluble Fraction), extracted as in [40 (link)] crude/purified synaptosomal proteins, was separated by SDS (Bio-Rad) polyacrylamide gel electrophoresis (ProtoGel, National Diagnostics). For SNARE complex detection, samples (referred to as unboiled) were kept at RT for 7 min before loading. Antibodies used were anti-p-JNK and-JNK (#9251 and #9252 both Cell Signalling, 1 : 1000), anti-Syntaxin-1 (#78539, Abcam), anti-Syntaxin-2 (#S0664, Sigma, 1 : 5000), Snap25 (Stressgen, 1 : 5000), anti-Syntaxin-1 (Abcam, 1 : 5000), anti-Vamp (#104 211, Synaptic Systems), anti-Synaptophysin (#S5768, Sigma), anti-PSD95 (#10011435, Cayman Chemicals, 1 : 1000), and anti-Actin (1501, Millipore, 1 : 5000). Blots were developed using ECL chemiluminescence system (ECL Western Blotting Substrate, Promega) and quantified by densitometry using ImageJ software.

The pET30a vector (constructed in our laboratory) was used to express N. farcinica Nfa34810 in E. coli. The suicide plasmid pk18mobsacB (constructed in our laboratory) was used to construct the nfa34810 deletion mutant. The following antibodies were used in this study: anti-p-Jnk and -JNK (4668; Cell Signaling), anti-p-ERK 1/2 and -ERK 1/2 (4370; Cell Signaling), anti-p-p38 and -p38 (4511; Cell Signaling), anti-p-p65 and -p65 (3033; Cell Signaling), anti-p-AKT and -AKT (4060; Cell Signaling), anti-β-actin (TransGen Biotech), anti-TLR4 (NB100-56727; Novus Biological, USA), anti-TLR2 (NB100-56726; Novus Biological, USA), and IgG_2A isotype control (MAB003; R&D Systems, USA). Human and mouse TNF ELISA kits were used in this study (BD, USA). The following reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA): p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059), JNK inhibitor (SP600125), and NF-κB inhibitor (BAY 11-7082). An endotoxin removal kit was purchased from GenScript (Nanjing, China).