Pa5 30604

The homogenate from the liver tissue was collected, and the lysate was used to lyse the hepatocytes for 30 min on ice. The BCA method was used to determine the protein concentration. The sample was added to 5× loading buffer by volume and heated in a water bath at 100 °C for 5 min. The hepatocytes were sonicated by SJIALAB for 2 min (10% ultrasound intensity) and centrifuged for 10 min to obtain the supernatant. The extracted protein was subjected to polyacrylamide gel electrophoresis (80–120 V), transferred to a PVDF membrane, sealed and incubated overnight at 4 °C with the addition of primary antibody of nNOS (3G6B10, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), eNOS (9D10, Invitrogen), iNOS (PA1036, Invitrogen), NF-κB (PA1186, Invitrogen), IκB-α (397700, Invitrogen), Mn-SOD (PA530604, Invitrogen), Cu/Zn-SOD (PA5270240, Invitrogen), CAT (PA259183, Invitrogen), HO-1 (ab68477, Abcam, Cambridge, MA, USA), Nrf2 (ab92946, Abcam), γ-GCS (sc-390811, Santa Cruz Biotechnology, Dallas, TX, USA), NQO1 (ab28947, Abcam) and β-action (MA5157739, Invitrogen). The protein was incubated overnight at 37 °C with the addition of a second antibody (A21241) for 2 h, and color detection and chemiluminescence imaging analysis system (Tanon 6200, ShanghaiTanon Technology Co., Ltd., Shanghai, China) were used to collect images [56 (link)].

Cell or tissue lysates with equal amounts of protein (40 μg) were mixed with loading dye, boiled for 5 min, separated on a denaturing 12.5-% SDS-polyacrylamide gel and transferred to Amersham™ Hybond™ 0.45 μm PVDF membrane (GE Healthcare, UK). The membrane was blocked in 5% dry milk or BSA in TBS buffer with 0.1% Tween (TBS-T) for 1 h and incubated overnight with antibodies against glutathione-S-transferase A3 (ab175246, Abcam), glutathione-S-transferase P1 (PA5-29601, Invitrogen), glutathione reductase (ab16801, Abcam), superoxide dismutase 2 (PA5-30604, Invitrogen), NAD(P)H quinone oxidoreductase 1 (PA5-19624, Invitrogen), pro-caspase 3 and active caspase 3 (ab13847, Abcam), or β-actin (4967, Cell Signaling) as a reference. Then, the membrane was washed twice with TBS-T and incubated with HRP-conjugated secondary antibody (ab6721, Abcam or sc-2005, Santa Cruz Biotechnology) at room temperature for 1 h, followed by several washings with TBS-T and deionized water. Protein bands were visualized by ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA) using chemiluminescence mode.

C17-SAMT was purified from the contaminated mussel extract (M. galloprovincialis) using a bio-guided approach. As described previously by Marrouchi et al. [17 (link)], HPLC purification coupled with mouse bioassays was carried out to obtain the purified toxin. Briefly, an Agilent 1100 series analyzer with a Hypersil ODS-2 column (C18, 4.6 m, 250 mm, 5 m, ThermoScientific, Illkirch, France) was used to calculate the toxin concentration. D-erythrosphinganine (C17-SPA) from Avanti Polar Lipids (Alabaster, AL, USA) and a certified C17-SPA (10 mg/mL) solution were used to calibrate, and peak areas were measured to calculate peak intensities. The purified fraction of the toxin was kept at −20 °C until it was analyzed.
Primary and secondary antibodies for HCA experiments were provided by Abcam (Cambridge, UK). Primary antibodies included rabbit polyclonal anti-histone H3 phospho S10 (ab5176), mouse monoclonal anti-γH2AX (ab26350), mouse monoclonal anti-phospho ATM S1981 (ab19304), rabbit polyclonal anti-NFκB (ab16502), and rabbit polyclonal anti-HO1 (ab13243). Secondary antibodies used were goat anti-rabbit IgG H&L, Alexa Fluor^® 488 (ab96891) and goat anti-mouse IgG H&L, Alexa Fluor^® 647 (ab96876). A rabbit polyclonal anti-SOD2 antibody (PA5-30604) was provided by Invitrogen (Invitrogen™, Illkirch, France).