2 16k centrifuge

E1, E2, EE2, and E3 (purity > 97%) were purchased from Sigma-Aldrich (Munich, Germany). Methanol and acetonitrile (HPLC grade) were obtained from Burdick & Jackson (Honeywell, Morristown, NJ, USA). NaOH (AR) and HCl (AR) were purchased from the Beijing Chemical Engineering Factory (Beijing, China). CaCl₂ was purchased from the Xilong Chemical Plant of Shantou, Guangdong. NaN₃ (AR) was obtained from the Tianjin Fuchen Chemical Reagents Factory. Sediments were collected from the Songhua River in the Jilin Province, China.
A 1200-HPLC (Agilent Company, Santa Clara, CA, USA), equipped with two model pumps (G1312A), an in-line degasser (G1322A), a column oven (G1316A), and a fluorescence detector (G1321A), was used for HPLC analysis. The injection loop volume was 20.0 µL, and a Zorbox SB-C18 column (250 mm × 4.6 mm; 5 µm) was used for the separations. A 2-16K centrifuge (Sigma, Munich, Germany), a FA-1004 analytical balance (Shanghai Hengping Science Instrument Company, Shanghai, China), and Milli-Q ultrapure water (Millipore, Billerica, MA, USA) were also used.

After euthanasia, brains were immediately removed and cortices dissected and snap-frozen for further studies. Brain cortices were then homogenized at 0–4 °C in lysis buffer, containing (in mM): 25 HEPES, 2 MgCl₂, 1 EDTA, 1 EGTA, pH 7.4, supplemented with 2 mM DTT, 100 µM PMSF and commercial protease and phosphatase inhibitors cocktails. The crude homogenate was centrifuged at 17,968× g for 10 min, at 4 °C in a Sigma 2–16K centrifuge to remove the nuclei, and the resulting supernatant was collected. Pellet was further resuspended in supplemented buffered solution and centrifuged again at 17,968× g for 10 min, at 4 °C. The supernatant was added to the previously obtained one and protein content determined by the Bio-Rad Protein Assay, according to the manufacturer’s instructions.

Mice were weighed and euthanized by decapitation, and brains were immediately removed. Brain cortices were immediately dissected and snap-frozen for further studies. Immediately before the experiments, brain cortices were homogenized at 0–4 °C in lysis buffer, containing (in mM) the following: 25 HEPES, 2 MgCl₂, 1 EDTA, 1 EGTA, (pH 7.4), supplemented with 2 mM DTT, 100 μM PMSF, and commercial protease and phosphatase inhibitors cocktails. The homogenate was centrifuged at 17,968 × g for 10 min, at 4 °C, in a Sigma 2–16 K centrifuge to remove the nuclei, and the resulting supernatant was collected. The pellet was further resuspended in supplemented buffered solution and centrifuged again at 17,968 × g for 10 min, at 4 °C. The supernatant was added to the previously obtained one and protein content was measured using Pierce BCA Assay Kit (Thermo Scientific, Rockford, IL, USA) according to manufacturer’s protocol.