Advanwash

2,4-Dinitrophenyl hydrazine (DNPH), paraquat (PQ), thiourea, 7-(diethylamino)-coumarin-3-carbohydrazide (CHH), Hoechst 33,258 (Hoechst), 2,7-dichlorofluorescin diacetate (DCFDA), tert-butyl methyl ether (MTBE), primary goat anti-DNP Ab and sodium, potassium, and ammonium salts were purchased from Sigma Aldrich GmbH (Taufkirchen, Germany). Formic acid was obtained from BiosolveBV (Valkenswaard, Netherlands). Dithiothreitol (DTT), urea, and ethanol were obtained from CarlRoth GmbH & Co. KG and chloroform was from Merck KGaA (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM/Ham's F12), fetal bovine serum (FBS), phosphate buffer saline (PBS), 7-aminoactinomysin-D (7-AAD), Trypan Blue (0.1%) and antibiotic (penicillin/streptomycin) solutions were obtained from Life Technologies GmbH (Darmstadt, Germany). Secondary rhodamine (TRITC) AffiniPure Rabbit Anti-Goat IgG (H+L) Ab and peroxidase-conjugated donkey anti-goat Abs were obtained from Jackson ImmunoResearch Laboratories, Inc. (Pennsylvania, United States). E06-monoclonalAb-TopFlour™ antibody was purchased from Avanti Polar Lipids, Inc. (Alabama, United States of America). Low fluorescent PVDF membranes, immunoblot blocking solution (AdvanBlock), immunoblot washing solution (AdvanWash) were purchased from Advansta (California, United States of America).

Proteins were dissolved in sample buffer (62.5 mmol/L TrisHCl, pH 6.8, 50 mmol/L DTT, 2% w/v SDS, 20% w/v glycerol, 0.2% w/v bromophenol blue) and separated by SDS-PAGE (10% T; BioRad mini protean III cell; BioRad Laboratories GmbH, München, Germany). Proteins were semidry blotted onto a low fluorescent polyvinylidene difluoride (PVDF) membrane (Trans-Blot Turbo Transfer System, BioRad Laboratories GmbH, München, Germany). Membranes were equilibrated (2 M HCl), derivatized with DNPH (1 g/L in 2 M HCl, 30 min, RT), washed with 2 M HCl (5 min) and methanol (5 min, 5 times). Membranes were blocked overnight (4 °C, Immunoblot Blocking solution; AdvanBlock, Advansta), incubated with goat anti-DNP Ab (1:10,000; in blocking buffer, 1 h, RT), and washed (Immunoblot Washing solution, AdvanWash, Advansta) before peroxidase-conjugated donkey anti-goat Ab (1:10,000, in blocking buffer) were added (1 h, RT). Membranes were visualized using WesternBright Sirius HRP substrate (Advansta) and imaged on a Fusion FX7 Imaging system (Peqlab Biotechnologie GmbH, Erlangen, Germany).