Kt5720

Transverse hippocampal slices (400 μm) were made using a standard mechanical tissue chopper and allowed to recover in oxygenated (95% O₂/5% CO₂) artificial cerebrospinal fluid (ACSF) at 32 °C for 1 h. cLTP was induced using 200 nM N-methyl-d-aspartate (NMDA; Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg²⁺ ACSF for 10 min followed by 0.1 μM rolipram + 50 μM forskolin (Cayman Chemical, Ann Arbor, MI, USA) in 0 Mg²⁺ ACSF for 15 min [29 (link),30 (link)]. ACSF lacking Mg²⁺ was used as NMDA receptors are normally blocked by such ions.
After 1 h of recovery, slices undergoing the cLTP induction protocol were incubated in ACSF with 25 μM β-lactone for 30 min or the following specific kinase inhibitors for 1 h; 20 μM U0126 (Cayman Chemical, Ann Arbor, MI, USA), 5 μM KT5720 (Cayman Chemical, Ann Arbor, MI, USA), or 5 μM KT5823 (Cayman Chemical, Ann Arbor, MI, USA); or the neddylation inhibitor MLN4924 (2 μM) (Cayman Chemical, Ann Arbor, MI, USA) followed by cLTP induction. Slices were then collected at different timepoints and processed for immunohistochemistry.

Antibodies against phospho-eNOS (Ser1177), phospho-AMPKα (Thr172), AMPKα, phospho-Akt (Ser473), phospho-p38 mitogen-activated protein kinase (p38 MAPK) (Thr180/Tyr182), p38 MAPK and cAMP response element binding protein (CREB) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phospho-CREB (Ser133) and eNOS (NOS3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phospho-acetyl CoA carboxylase (ACC) (Ser79) antibody was purchased from Millipore (Billerica, MA). An anti-myc tag antibody was purchased from Upstate Biotechnology (Lake Placid, NY) and an anti-HA tag antibody was purchased from Roche (Basel, Switzerland).
Wortmannin, LY294002, and KT5720 were purchased from Cayman Chemical Company (Ann Arbor, MI). H89 and GSK3787 were purchased from Tocris Bioscience (Bristol, UK). GW9662 was purchased from Wako Pure Chemical (Osaka, Japan). SB202190 was purchased from Calbiochem (Darmstadt, Germany).
Valsartan and irbesartan were purchased from Vijayasri Chemicals (Andhra Pradesh, India). Telmisartan was provided by Boehringer Ingelheim (Ingelheim, Germany).

To confirm the role of TLR-2 activation in downregulating SERT activity following bacterial infection, we blocked TLR-2 receptors using selective human TLR-2 antagonist (Novus Biologicals, Littleton, CO) to rule out the role of other TLRs in regulating SERT function. Caco-2 monolayers were pre-incubated with 5 ug/mL of TLR2 inhibitor for 30 min prior to MAP infection and 5-HT treatment. We also assessed TLR-2 intracellular signaling pathways in depth by pre-incubating Caco-2 monolayers with selective blockers of ERK (40 uM PD98059; Cayman Chemical, Ann Arbor, MI), cAMP/PKA (1 uM KT5720; Cayman Chemical, Ann Arbor, MI) and p38 MAPK (1 uM SB220025; Sigma Aldrich, St. Louis, MO) for 30 min prior to MAP infection and 5-HT treatment (500 ng/mL).