Rabbit anti cysteine sulfenic acid

Protein lysates were separated on 4–12% Bis-Tris gels and transferred to nitrocellulose membranes. The membranes were blocked for 60 min at room temperature in 5% milk in TBS-T (0.05% Tween-20, pH 7.4), washed with TBS-T and then incubated with primary antibody [rabbit anti-Cav-1 (Abcam, Cambridge, MA), mouse anti-Cav-1 (BD Technologies, Research Triangle Park, NC), rabbit anti-β-actin (Cell Signaling Technologies, Boston, MA), rabbit anti-MnSOD(Abcam), rabbit anti-AMPK pThr-172 (Abcam), rabbit anti-Nrf2(Santa Cruz), mouse anti-Keap1 (Abcam), rabbit anti-Cysteine Sulfenic Acid (EMD Millipore, Billerica, MA)] in TBS-T overnight at 4°C. Antibodies were diluted 1:1000 unless stated otherwise. After 3 washes, membranes were incubated with the infrared secondary antibody to anti-rabbit/mouse, 1:5,000 in TBS-T and incubated for 2 hours at room temperature. After 3 washes, protein signals were analyzed by Odyssey CLx Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).

Freshly harvested or after-ripened seeds were imbibed for 24 h at 25 °C, either with water or with ascorbate and copper (ascorbate 10 mM plus CuSO₄ 10 µM) to trigger cysteine oxidation to sulfenic acid (RSOH) by ROS [86 (link)]. RSOH in seed proteins was detected after chemical derivatization with 5,5-dimethyl-1,3-cyclohexanedione (dimedone) forming a stable thioether adduct that could be immunodetected. Protein samples (50 µg) were separated by 4–20% SDS-PAGE. Following electrophoresis, proteins were transferred onto a PVDF membrane and detected using 1:10,000 dilution of a rabbit anti-cysteine sulfenic acid (Merck Millipore, Guyancourt, France) antibody and 1:20,000 diluted anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare, Tremblay-en-France, France), and finally, oxidized proteins were visualized by chemiluminescence (GE Healthcare, Tremblay-en-France, France). Seed protein thiols and carbonyls were detected as previously described [31 (link),87 (link)].