D erythrosphinganine c17 spa

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).

The C17-SAMT was extracted from contaminated mussels M. galloprovincialis as described previously by Marrouchi et al. (2013) [19 (link)], with a purification process using an HPLC bio-guided approach (Figure S1). The toxin concentration was estimated using an Agilent 1100 series analyzer with a Hypersil ODS-2 column (C18, 4.6 µm × 250 mm, 5 µm, ThermoScientific, Illkirch, France). To calibrate, a certified C17-SPA (10 mg/mL) solution was employed, and peak areas were measured to determine peak intensities. D-erythro-sphinganine (C17-SPA) from Avanti Polar Lipids (Alabaster, AL, USA). All other chemicals used were of the highest grade commercially available.