Na2s4

After preincubation for 1 hr at 37 °C, 300 μl suspensions of brain cells were incubated for 15 min at 37 °C in the presence of 500 μM 3 MP (Sigma-Aldrich), or 500 μM Na₂S (Wako). After the exposure to 3 MP or Na₂S the suspensions of brain cells were centrifuged at 100 x  g for 30 sec, and the supernatant was removed. Cells were suspended in 300 μl basic medium containing 14 mM glucose in HBSS with Ca²⁺ and Mg²⁺, and removed the supernatant after centrifugation at 100 × g for 30 sec. This step was repeated three times to wash out 3 MP. Cells were sonicated in BHM solution and centrifuged at 15,000 × g for 10 min at room temperature. The supernatant was incubated in the presence of 125 mM CHES (pH 8.4) and 2 mM monobromobimane for 20 min at room temperature, and then acetic acid was added to the final concentration of 3% and incubated for 15 min on ice. The resulting reaction mixture was centrifuged at 15,000 × g for 10 min, and the supernatant was analyzed by LC-FL and LC-MS/MS. Na₂S₂, Na₂S₃, and Na₂S₄ for standard were obtained from Dojindo (Kumamoto, Japan).

SSP4 (3′,6′-di(O-thiosalicyl)fluorescein), Na₂S₂, Na₂S₃, and Na₂S₄ were purchased from Dojindo molecular Technologies Inc. (Rockville, MD, USA). MnTBAP, which is also a Mn porphyrin, but lacks appreciable SOD-mimetic activity [26 (link)] was purchased from Sigma–Aldrich (St. Louis, MO, USA), other MnPs were synthesized as previously reported [27 (link),28 (link)]. All other chemicals were purchased either from Sigma-Aldrich or ThermoFisher Scientific (Grand Island, NY, USA). Please note that we use H₂S to denote the total sulfide added (sum of H₂S + HS⁻) usually derived from Na₂S. Also, while S²⁻ is often thought as part of the H₂S + HS⁻ equilibrium, it does not exist under these conditions as the pK > 12 [29 (link)]. Phosphate buffer (PBS; in mM): 137 NaCl, 2.7 KCl, 8 Na₂HPO₄, 2 NaH₂PO₄. pH was adjusted with 10 mM HCl or NaOH to pH 7.4.

Dimethyl sulfoxide (DMSO), 1,4-NQ (98% purity determined by gas chromatography) and anti-GAPDH antibody were from Wako Pure Chemical Industries (Osaka, Japan), Tokyo Chemical Industries (Tokyo, Japan) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Biotin-PEAC₅-maleimide (BPM) and Na₂S₄ were from Dojindo Laboratories (Kumamoto, Japan). Dynabeads M-280-sheep anti-rabbit immunoglobulin G (IgG) was from Invitrogen (Carlsbad, CA, USA). Anti-Akt, anti-CREB, anti-phosphorylated Akt (Ser473), anti-phosphorylated CREB (Ser133), horseradish peroxidase (HRP)-conjugated anti-biotin antibodies, anti-rabbit antibodies and anti-mouse IgG secondary antibodies were from Cell Signaling Technology (Beverly, MA, USA). Escherichia coli BL21 cells and trypsin were from Promega Co. (Madison, WI, USA). Glutathione 4B Sepharose was from GE Healthcare (Chicago, IL, USA). All other reagents were of the highest purity available.