Ammonium molybdate

The extraction solvents used, as well as specific chemical reagents, included PBS, Na₂CO₃ ≥ 99.5% (Cas No.497-19-8), AlCl₃, Folin–Ciocalteu (Cat. No 109001), ferric chloride (Cas No.7705-08-0), potassium acetate, quercetin (Cas No.117-39-5), gallic acid (Cas No. 149-91-7), vanillin (Cas No. 121-33-5), sulfuric acid, hydrochloric acid (HCl), sodium phosphate, ammonium molybdate, and DPPH (2,2-diphenyl-1-picrylhydrazyl, Cas No. 1898-66-4), (Sigma-Aldrich, St. Louis, MO, USA). HPLC-grade solvents were procured from Merck (Darmstadt, Germany). Water purification was carried out using the Direct-Q UV system from Millipore, (St. Louis, MO, USA). High-purity standard compounds were used for HPLC, specifically, gallic acid, chlorogenic acid (with 99% HPLC purity), and rutin (with 99% HPLC purity) (Sigma, St. Louis, MO, USA). Various culture mediums were used, including nutrient broth, Baird-Parker agar, TBX agar, XLD agar, Palcam agar, and Muller Hinton agar (Oxoid Ltd., Basingstoke, Hampshire, UK). All chemicals and reagents used in this study met analytical grade standards.

Sulfuric acid, sodium hydroxide, acetonitrile (Ultra high performance liquid chromatography (UHPLC) grade), ethylenediaminetetraacetic acid (EDTA), sodium borate, and hydrochloric acid were obtained from Panreac (Castelar del Vallés, Barcelona, Spain). Sodium acetate (anhydrous), trimethylamine (TEA), phosphoric acid, ammonium molybdate, and methyl cellulose were purchased from Sigma-Aldrich (Darmstadt, Germany). Standard of amino acids (L-arginine (Arg), L-alanine (Ala), L-asparagine (Asn), L-aspartic acid (Asp), glycine (Gly), L-glutamic acid (Glu), L-glutamine (Gln), L-histidine (His), L-isoLeucine (Ileu), Leucine (Leu), L-Lysine (Lys), L-norvaline (Nor), L-phenylalanine (Phe), L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Trp), L-tyrosine (Tyr), and L-valine (Val)) were purchased from Sigma-Aldrich (Steinheim, Germany). All the other chemicals and reagents used were of analytical grade.

To remove ATP, actin monomers in G-buffer were incubated with Bio-Rad AG1-X2 100–200 mesh anion exchange resin (Dowex) (Bio-Rad, 1401241) rotating at 4°C for 5 min, followed by low-speed centrifugation to remove resin. Actin filaments (20 μM) were polymerized in 50 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Hepes pH 7.4 for 1 hr at 23°C. Drp1 was diluted in 150 mM KCl, 1 mM MgCl₂, 1 mM EGTA, 10 mM Hepes pH 7.4, then centrifuged to remove aggregates as described above.
Drp1 (1.3 μM) was mixed with actin filaments (1 μM), and the final ionic strength was adjusted to the equivalent of 75 mM KCl using 4 M KCl stock. Samples were incubated at 37°C for 5 min. At this point, GTP was added to a final concentration of 250 μM to start reactions at 37°C. Reactions were quenched at various time points by mixing 20 μL of sample with 5 μL of 125 mM EDTA in a clear flat-bottomed 96-well plate (Greiner). Six time points were acquired for each condition. Inorganic phosphate was determined by addition of 150 μl of Malachite green solution (1 mM Malachite green (Sigma Aldrich, 2290105–100 g), 10 mM ammonium molybdate ([Sigma Aldrich, A7302–100 g] in 1N HCl) to 25 μl quenched reactions. Absorbance at 650 nm was measured with a 96-well fluorescence plate reader (TECAN Infinite M1000, Mannedorf, Switzerland). GTP hydrolysis rates were determined by plotting phosphate concentration as a function of time.

The LWO material was prepared by a modified Pechini method^{27 (link)}. Lanthanum nitrate hexahydrate (Sigma Aldrich), ammonium tungstate (Sigma Aldrich) and ammonium molybdate (Sigma Aldrich) were used as the precursors. The complexation reaction was performed as shown in^{11 (link)}. The mixtures were calcined at 900 °C in air to remove the organic constituents and form an oxide. Afterwards, 1 gram of the powder was pressed to pellets with a diameter of 20 mm. The final sintering step was performed at 1500 °C for 12 h with heating rates of 5 K/min to obtain high densification and complete phase formation. XRD measurements were performed with a Bruker D4 X-ray diffractometer. The Mo concentration was varied between 0 and 35%, meaning that up to 35% of the W was substituted by Mo. Higher Mo concentrations appeared to be not stable after storage in air for several days.