Complete tablets easypack

Repagermanium (lot. 006316 A, purity > 99.9%), the polymer of THGP, was produced at the Hakodate plant of the Asai Germanium Research Institute Co., Ltd. (Hokkaido, Japan), and Bis(2-carboxyethylgermanium(IV) sesquioxide) was purchased from Sigma-Aldrich (Missouri, USA). Potassium bromide (KBr) and D₂O were purchased from Kanto Chemical, Co., Inc (Tokyo, Japan). NHDFs and a Fibroblast Growth Medium 2 Kit were purchased from Takara Bio (Shiga, Japan). Xanthine oxidase (XOD from buttermilk) was purchased from Oriental Yeast Co., Ltd. (Tokyo, Japan). Hypoxanthine (HPX), hydrogen peroxide and 4% paraformaldehyde phosphate buffer solution were purchased from Wako Pure Chemical Industrial Co., Ltd. (Osaka, Japan). Cellstain-PI solution and Hoechst 33342 solution were purchased from Dojindo Laboratories Co., (Kumamoto, Japan). A complete tablets EASY pack was purchased from Roche Diagnostics GmbH (Mannheim, Germany). NR4A2 and β-actin antibodies were purchased from Santa Cruz Biotechnology (California, USA) and Abcam (Cambridge, England). Secondary antibodies, Goat Anti-Mouse IgG H&L (HRP) and Donkey Anti-Goat IgG H&L (HRP) were purchased from Abcam. All reagents used in this study were of special or molecular biological research grade.

Protein lysates were generated from cell pellets using Magic Mix (48% urea, 15 mM Tris pH 7.5, 8.7% Glycerin, 1% SDS, 143 mM β-mercaptoethanol) containing protease and phosphatase inhibitors (cOmplete Tablets easypack, PhosSTOP easypack, Roche) and transferred to a QIAshredder column (QIAGEN). After centrifugation at 12,000g for 2 min, the solution was transferred into a fresh tube and frozen at −80°C until needed. The concentration was not measured. SDS gel electrophoresis was used to separate proteins by their size. Proteins were transferred to a PVDF membrane by using the Trans Blot Turbo Transfer Pack (Bio-Rad). Membranes were incubated for 1 h with blocking buffer (PBS, 0.1% Tween, 5% milk), followed by overnight incubation with primary antibody diluted in blocking buffer. Membranes were washed three times for 10 min with PBS-T (PBS, 0.1% Tween), incubated for 1 h with secondary antibody diluted in blocking buffer, and washed three times for 10 min with PBS-T (PBS, 0.1% Tween). Membranes were exposed using the Western Lightning Plus-ECL (Perkin Elmer), and imaging was performed by using ChemiDoc Imaging System (Bio-Rad). Images were prepared and analyzed using the Image Lab software (Bio-Rad). Mouse monoclonal anti-β-ACTIN (A2066-200UL; 1:2,000; Sigma-Aldrich), rabbit polyclonal anti-MID1 C-terminal (NBP1-26612; 1:500; Novus).

Tissues were homogenized in Qiazol (Qiagen, Hilden, Germany, 79306) using Magnalyser beads (PeqLab, Radnor, United States, 412-0201) at 6.500 rpm for 20 s and two runs with the TissueLyser (Qiagen, Hilden, Germany). Samples were cooled with short taps in N₂ between the runs and incubated for 5 min at room temperature. RNA was isolated with PeqGOLD total RNA kit (Peqlab, Radnor, United States, 12-6634) according to the manuals. Sample purification and concentration was quantified with NanoDrop® ND-1000 (Peqlab, Radnor, United States). For western blotting experiments, tissues were homogenized with Magnalyser beads in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 0.1% SDS, 0.5% Na-deoxycholate, 1% NP-40, adjusted to pH 7.2 — 7.4) supplemented with PIC (complete Tablets EASYpack, Roche, Basel, Switzeland, 04693116001) and PhosStop (Roche, Basel, Switzeland, 04906837001), incubated on ice for 20 min, and centrifuged at 15.000 x g for 15 min. The protein concentration of cleared supernatants was analyzed with a bicinchoninic acid assay kit (BCA, Thermo Fisher Scientific, Waltham, MAs, United States).

Lysis buffers were supplemented with 1 × protease inhibitor cocktail (PIC) (cOmplete Tablets EASYpack, Roche, 04693116001) and 1 × PhosSTOP (Roche, 04906837001). Total protein lysates were obtained after washing the cells with PBS, and application of the respective buffers provided in each protocol segment.

Subjects were requested not to eat or drink for at least 30 min before collection. All patients rinsed their mouths with 10 mL of water for 30 s. After 10 min, whole unstimulated saliva collection began. Between 8:00 a.m. and 11:00 a.m., each participant expectorated continuously in a 50 mL sterile propylene tube for a period of 5 min. Additionally, we collected the salivary pellicle deposited on lesions and the contralateral anatomical equivalent of the unaffected oral mucosa using cotton swabs. Immediately after collection, samples were deposited on ice and a protease inhibitor cocktail (cOmplete Tablets EASYpack, Roche)^{26 (link)}. We then centrifuged the samples at 14,000×g at 4 °C for 20 min to eliminate undissolved components and cellular detritus^{27 (link)}. The total protein concentration in the supernatants was determined using the BCA Protein Assay (Thermo Scientific). The samples were stored at − 80 °C for further processing.

The quantification of H₂O₂ was based on a ferrous oxidation/xylenol orange assay using the Pierce Quantitative Peroxide assay according to the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, 50 mg of the mouse kidney tissue was weighed and homogenized in homogenization buffer with 500 nM Tris-HCl (pH 7.0), 1% protein inhibitor cocktail (cOmplete Tablets EASYpack, Roche, Mannheim, Germany), and 1% NP-40 (KO: 6; WT: 6). After centrifugation at 9500× g and 4 °C for 15 min, an aliquot of the supernatant was transferred to a tube and protein was measured according to Lowry et al. [69 (link)], using bovine serum albumin as the standard. After the sample protein concentration was adjusted to 264 mg/mL, the hydrogen peroxide concentration was determined. Test tubes containing 100 μL of the sample solution and 1 mL of the working reagent solution were maintained at 30 °C for 30 min and then immediately measured at a wavelength of 560 nm using a spectrometer. Absorbance values were calibrated to a standard curve generated using a known concentration of H₂O₂.

DOC was obtained from Aventis Pharmaceuticals Inc. (Bridgewater, NJ). VCR and N-acetylcysteine (NAC, A-7250) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK was purchased from Bachem (Torrance, CA, USA). U0126 and SB203580 were purchased from Cell Signaling (Danvers, MA, USA). Curcumin was obtained from Cayman Chemical (Ann Arbor, MI, USA). PhosSTOP and cOmplete Tablets EASYpack were purchased from Roche (Basel, Switzerland).