U0126

3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), sGC inhibitor ODQ, non-competitive selective PDE inhibitor IBMX, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PKA inhibitor H89 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The sGC activator BAY 41-2272 and NF-κB inhibitor Ro 106-9920 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The extracellular regulated MAP kinase (ERK) inhibitor U0126, p38 MAP kinases inhibitor SB202190, c-Jun N-terminal kinases (JNK) inhibitor SP600125, HIF-1α inhibitor CAY10585, p38 activator anisomycin, and phosphatidylinositol-3-kinases (PI3K) inhibitor wortmannin were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Anti-LC3B, anti-Bcl-xL, anti-Atg16L1, anti-Rac1, anti-Rho1, anti-Cdc42, anti-phospho-ERK, anti-EEA1 and anti-LAMP1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibodies reactive with LRP1 (11H4) were a kind gift from Dr. Strickland, University of Maryland School of Medicine, Baltimore. Fibronectin (EP5) and ubiquitin (P4D1) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); anti-Cx43, anti-ERK, anti-Bcl-2 and anti-RPTPβ antibodies were from BD Biosciences (Tokyo, Japan); anti-multi ubiquitin monoclonal antibody (FK1) was from MBL (Nagoya, Japan); anti-GAPDH antibody was from GeneTex (Irvine, CA, USA) and anti-LC3 (clone 1703) antibody was from Cosmo Bio (Tokyo, Japan). Anti-RPTPα rabbit polyclonal antibodies for immunoblotting were provided by Dr. Jan Sap; anti-α-tubulin and anti-FLAG M2 antibodies, N-acetyl-l-cysteine (NAC) and ammonium chloride were from Sigma Aldrich (St. Louis, MO, USA); ProLong Gold Antifade Reagent with DAPI and DIDS were from Invitrogen (Eugene, OR, USA); an ERK inhibitor, U0126, was from Cayman Chemical (Ann Arbor, MI, USA); Rac1 inhibitor, NSC23766 was from Wako Pure Chemical Industries (Osaka, Japan); and GSH was from Merck (Darmstadt, Germany).

MTZ (MilliporeSigma, M3761-25G) was dissolved in system water at a concentration of 5 mM and 0.5% DMSO. Adult zebrafish were exposed to MTZ for 16 hours at ambient temperature in the dark. APAP (MilliporeSigma, A7085-100G) was dissolved in system water at a concentration of 15 mM. EtOH (MilliporeSigma, EX0276-3) was dissolved in system water at a concentration of 1%. Adult zebrafish were exposed to APAP or EtOH for 24 hours at ambient temperature in the dark. Stock solutions for AG1478 (Selleck Chemicals, S2728), U0126 (Cayman Chemical Company, 70970), LY290002 (Selleck Chemicals, S1105), and Rapamycin (Selleck Chemicals, AY-22989) were made by dissolution in DMSO at a concentration of 10 μM, 20 μM, 10 μM, and 10 μM, respectively. These chemicals were diluted 1:1,000 in system water to make a working solution. Adult zebrafish were immersed in the working solution. Working solution was changed daily. 4-OHT (Hello Bio, HB2508) was dissolved in DMSO at a concentration of 10 mM, vortexed continuously for 15 minutes, and stored at –20°C. Prior to use, an aliquot of 4-OHT was incubated at 65°C for 10 minutes. Triple-transgenic larvae were exposed to 4-OHT between 2 and 3 days after fertilization to induce recombination.

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.

MeT-5A, NCI-H2052, NCI-H2452, NCI-H28 and MSTO-211H cells were provided by ATCC and cultured in RPMI (Thermo Scientific, Waltham, MA, USA), supplemented with 10% FBS (R&D Systems, Minneapolis, MN, USA) and 1% L-glutamine (Thermo Scientific Scientific).
The MEK1/2 inhibitor U0126, the FAK inhibitor PF-573228, the EGFR inhibitor gefitinib and nocodazole were from Cayman Chemicals (Ann Arbor, MI, USA) and the AKT inhibitor AKTi VIII form Calbiochem (San Diego, CA, USA). His-tag human recombinant progranulin was prepared as previously described [34 (link)].