Stock

solutions of esterase (E3019; Sigma Inc., St. Louis, MO, USA), horseradish peroxidase (Sigma P8125), and catalase were prepared in DI water daily. The reaction mixture was inverted to mix and incubated for 3 min at room temperature before reading on a Fluoromax 2 fluorometer (HORIBA Ixazomib cost Jobin Yvon Inc., Edison, NJ, USA). Excitation and emission were 488 and 525 nm, respectively. The experiment was conducted under low light conditions to avoid photooxidation of DCFH during sample incubation. Fluorescence readings in counts per min were converted to concentration of strong oxidants by comparison to a standard curve generated daily using H2O2. The concentration of strong oxidants was converted to the number of moles of oxidants released per gram of algal fresh weight (nm oxidants · g−1 FW) and strong oxidant release immediately upon wounding was determined by subtracting the amount of oxidants measured in

the seawater medium before wounding from those measured in the medium 1 min after wounding. The ability of 500 U catalase to decompose 1 M H2O2 in ice-cold seawater selleck products was verified each day by measuring DCFH fluorescence of a dilution of 30% H2O2 (Sigma 216763) made in ice-cold SFSW as above with and without the addition of 500 U catalase from the fresh catalase stock solution. In order to look at the relative timeline of the oxidative burst, we sampled oxidant release in the seawater medium over the course of 65 min after the wounding of three species (A. mirabilis, P. decipiens, and T. antarcticus). Samples were wounded at t = 5 min and oxidants were measured as above, with and without the addition of 500 U catalase, in the medium of each species (n = 3) at times 0, 20, 40, and 70 min. To determine whether RNS were a component of the wound-induced oxidative burst, paired samples of A. mirabilis, D. anceps, P. decipiens, and T. antarcticus were flash frozen in liquid nitrogen 30–60 s after punching with a sterile pipette tip. Protein was extracted from each

sample using a plant total protein extraction kit (Sigma PE0230) and quantified using a Bradford-based check details protein microassay (Bio-Rad protein assay kit II #500-0002), both according to the manufacturer’s protocol. For comparison, protein was also extracted from samples of Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl et G.W. Saunders collected from the dock at Friday Harbor Laboratories (University of Washington, Friday Harbor, WA 98250, USA) in August 2012 and exposed to peroxynitrite (ONOO−, Cayman Chemical 81565, Ann Arbor, MI, USA), an RNS and a strong nitrating agent. Protein samples were stored at −80°C until use. Nitrated proteins were detected using 1-D 10% SDS polyacrylamide gel electrophoresis (PAGE) with subsequent transfer to PVDF membrane and immunoblotting. 20–40 μg of protein were loaded for 1-D PAGE depending on the species, with equal amounts loaded for each replicate within a species (n = 6 for P.