Sodium molybdate

Methanol (HPLC or MS grade), ethanol, formic acid, acetic acid, hydrogen peroxide (30% solution), trichloroacetic acid (TCA), Folin–Ciocalteau’s reagent, 2-deoxy-D-ribose, sodium nitrite, sodium nitroprusside, potassium persulfate, sodium molybdate, aluminum chloride (III), ethylenediaminetetraacetic acid (EDTA), ascorbic acid, sulfanilamide, N-(1-naphthyl)ethylenediamine dihydrochloride, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), thiobarbituric acid (TBA), and standards of rosmarinic acid, catechin, luteolin, apigenin, and ursolic acid were purchased from Sigma-Aldrich/Merck (Algés, Portugal). Caffeic acid was obtained from Extrasynthese^® (Genay, France). Oleanolic acid was obtained from Santa Cruz Biotechnology Inc. (Frilabo; Porto, Portugal). Dulbecco’s Modified Eagle Medium (DMEM), sodium pyruvate, penicillin, streptomycin, versene, L-glutamine, trypsin-EDTA, and foetal bovine serum (FBS) were obtained from Gibco (Alfagene, Invitrogen, Portugal). Alamar Blue^® was obtained from Invitrogen, Life-Technologies (Porto, Portugal).

MV, BV, MB, NBT, PMS, TA, FAD, sodium molybdate, and sodium dithionite were purchased from Sigma-Aldrich (St. Louis, MO, USA). BugBuster^® and Ni-NTA resin were bought from Novagen (Billerica, MA, USA) and Qiagen (Valencia, CA, USA), respectively. Pfu polymerase, EcoRI, HindIII, and DpnI were purchased from Enzymonics (Daejeon, Korea). As a CO-containing waste gas from the steelmaking process, BFG was prepared from Seongkwang (Seoul, Korea). Unless otherwise stated, all chemicals were used at the highest grade available without further purification.

NS-398 was purchased from Alexis Deutschland GmbH (Grünberg, Germany). Aprotinin, Δ-glycerophosphate, ethylenediaminetetraacetic acid (EDTA), leupeptin, lovastatin lactone, luminal, mevastatin, p-coumaric acid, phenylmethylsulfonyl fluoride (PMSF), (R)-mevalonic acid lithium salt, sodium molybdate and sodium orthovanadate were obtained from Sigma-Aldrich (Taufkirchen, Germany). 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was from Ferak (Berlin, Germany). Dimethyl sulfoxide (DMSO), dithiothreitol (DTT), glycerol, p-nitrophenylphosphate, sodium chloride, sodium dodecylsulfate (SDS) and sodium fluoride were from AppliChem (Darmstadt, Germany) and GW9662 and Nonidet^® P-40 from Enzo Life Sciences (Lörrach, Germany). Lovastatin hydroxy acid, sodium salt, was provided from Toronto Research Chemical (Toronto, Canada) and Triton^® X-100, acetonitrile (LC-MS grade) and trifluoroacetic acid (analytical grade) from Roth (Karlsruhe, Germany). Penicillin-streptomycin was from Invitrogen (Darmstadt, Germany). Dulbecco's Modified Eagle's medium (DMEM) with 4 mM L-glutamine and 4.5 g/L glucose was from Lonza (Cologne, Germany). Phosphate-buffered saline (PBS) and fetal calf serum (FCS) were obtained from PAN Biotech (Aidenbach, Germany).

After 24 h of incubation, all the plates were placed on ice. The medium was removed, and the cells were washed twice with ice cold PBS. After washing, 600 μl of ice-cold lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with protease and phosphatase inhibitors: PMSF, cOmplete Mini EDTA free Protease Inhibitor Coctail (Roche), Sodium molybdate (Sigma-Aldrich) and Sodium Orthovanadate (Sigma-Aldrich) was added. After 5 min of incubation with periodic mixing, the lysates were transferred to a microcentrifuge tube and centrifugated at 13000 x g for 15 min at 4 °C. The obtained soluble fractions were incubated for 2 h on ice with 20 μl of EZview™ Red ANTI-FLAG M2 Affinity gel (Sigma-Aldrich) pre-equilibrated with TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4). After incubation, the gel was washed four times with 500 μl of TBS buffer. Finally, the gel was incubated with 100 μl of elution buffer (TBS buffer supplemented with 100 μg/ml FLAG peptide, Sigma-Aldrich) for 30 min on ice. The eluted proteins were collected for further analysis.

Chemicals used in this study included pyrrole (Py) monomer (98%), sodium salicylate (99.5%), sodium molybdate (98%), and sodium chloride (99.5%), all purchased from Sigma-Aldrich. Pyrrole monomer was purified by distillation prior to use and stored at a low temperature, protected from light. Graphene oxide (GO) was obtained from the Vietnam Academy of Science and Technology.
Commercial low-carbon steel was purchased from TISCO Company, Thai Nguyen, Vietnam. The composition (wt%) was 0.10% C, 0.5 Mn, 0.04 P, and 0.05 S.

Graphite was purchased from Alfa Aesar (325 mesh, Thermo Fisher Scientific, Waltham, MA, USA). Bombyx mori cocoons were provided by the Miaoli District Agricultural Research and Extension Station, Council of Agriculture, Executive Yuan of the ROC. H₂O₂ (30%) was purchased from Merck Millipore (Billerica, MA, USA). HRP (Type VI, ≥250 units/mg solid, MW ~ 44.1 kDa), dopamine hydrochloride (MW = 153.18 Da), 1,10-phenanthroline (MW = 180.2 Da), potassium permanganate (MW = 158.03 Da), sodium nitrate (MW = 84.99 Da), sodium nitrite (MW = 68.99 Da), and sodium molybdate (MW = 205.92 Da) were bought from Sigma Aldrich (St. Louis, MO, USA). An MTS assay kit for in the biocompatibility test was purchased from Promega (Madison, WI, USA). The Bradford reagent for the protein assay was purchased from Bio-RAD Corp (Hercules, CA, USA).