Sephadex g 50

Genomic DNA for genotyping was isolated from peripheral blood of 145 pigs using Blood Mini kit (A&A Biotechnology). PCR reactions were performed using primers overlapping known exonic SNPs that were used as reporter SNPs (rSNP) to quantify allele-specific expression: rs342258309 (A > G) in PPARA, rs319172675 (A > G) in PPARG, rs45430917 (A > T) in PPARGC1A, and rs712230598 (C > T) in SREBF1. For SREBF1, genotypes of some pigs were retrieved from our previous study [44 (link)]. Sequences of PCR primers are shown in Additional file 6. Prior to Sanger sequencing amplicons were purified using Exonuclease I (Thermo Scientific) and Alkaline Phosphatase (Thermo Scientific) and sequencing PCR performed using BigDye Terminator v.3 Cycle Sequencing kit (Thermo Scientific). Sequencing products were filtered on Sephadex G-50 (Sigma-Aldrich) and separated by capillary electrophoresis on 3130 Genetic Analyzer (Applied Biosystems).

We purified the endpoint RT-PCR products with Sephadex^® G-100 (Sigma-Aldrich, St. Louis, MO, USA) columns by centrifugation at 770×g for 3 min and then prepared the sequencing reactions with the BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). The sequencing reactions had a final volume of 10 μl containing 1 μl BigDye^® Terminator v3.1 Ready Reaction Mix, 1.5 μl 5× Sequencing Buffer, 0.2 μM of either the forward (FS778) or the reverse (cFD2) primer and 3 to 10 ng cDNA. The temperature profiles were as follows: 1 min at 96 °C, followed by 25 cycles of 10 s at 96 °C, 5 s at 50 °C and 4 min at 60 °C. We then purified the sequencing products with Sephadex^® G-50 (Sigma-Aldrich) columns by centrifugation at 770×g for 3 min and mixing with 5 μl of HiDi Formamide (Applied Biosystems) and sequenced the prepared cDNA in both directions on an Applied Biosystems 3500 Genetic Analyser (Applied Biosystems). We examined the sequences with the MEGA6 Software [37 (link)] and compared them against the BLAST Nucleotide database [38 (link), 39 ].

TSK gel G2000 SWXL and Sephadex G50 (50-150 μm) were obtained from Sigma-Aldrich Corp., Merck, St. Louis, MO, USA. Pepsin (300 000 U/g), papain (800 000 U/g), trypsin (1:250 U/g), cholesterol, oleic acid, and bicinchoninic acid protein assay kit were obtained from Guangzhou Qiyun Biotech Co., Ltd., Guangzhou, PR China. Alcalase 2.4 L (2.4 U/g) and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) were from Sigma-Aldrich Corp., Merck. Dulbecco's modified eagle medium (DMEM), fetal bovine serum, bovine serum albumin (BSA), pravastatin, the alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD) testing kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, PR China. All other reagents were of analytical grade.

We carried out a multispacer sequence typing (MST) technique on all fecal specimens positive by PCR-sequencing as previously described in our laboratory [23 (link),45 (link)]. PCRs were realized in a 2720 Thermal Cycler (Applied Biosystems, Foster City, California, USA) and followed all the steps described for standard PCR used for the molecular analysis of fecal specimens. Negative controls consisting of PCR mixture without DNA template were included in each PCR run. All PCR products were sequenced in both directions using the same primers as used for PCRs in a 2720 Thermal Cycler (Applied Biosystems) with an initial 1-min denaturation step at 96 °C, followed by 25 cycles denaturation for 10 s each at 96 °C, a 20 s annealing step at 50 °C, and a 4-min extension step at 60 °C. Sequencing products were purified using the MultiScreen 96-well plates Millipore (Merck, Molsheim, France), containing 5% of Sephadex G-50 (Sigma-Aldrich), and sequences were analyzed on an ABI PRISM 31309 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) and edited using the ChromasPro software (version 1.42; Technelysium Pty Ltd., Tewantin, Australia). For each intergenic spacer, a spacer type (ST) was defined as a sequence exhibiting unique genetic polymorphism (SNPs and indels). MST genotypes were defined as a unique combination of the four spacer sequences [23 (link),45 (link)].

Crude Her–TAT_(1–5) conjugates were purified by Sephadex-G50 (Sigma-Aldrich) size exclusion chromatography in a 1 mL column, eluting with 100 μL fractions of PBS (pH 7.2). Her–TAT_(1–5) conjugates were eluted between fractions 8–12. Fractions were combined to achieve the desired DOL_TAT value of 0–5.

Inorganic reagents including magnesium chloride hexahydrate (MgCl₂·6H₂O), aluminum chloride (AlCl₃), and sodium hydroxide (NaOH), used for the layered double hydroxide (LDH) preparation, were purchased from Sigma Aldrich (St. Louis, MO, USA). RES, Sephadex G-50, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), and all solvents were also obtained from Sigma Aldrich and used without further purification. Milli-Q ultrapure water (18.2 MΩ cm) was used throughout the study and adequately purged with N₂ before use. The human lung adenocarcinoma epithelial cell line (A549) was obtained from American Type Culture Collection (ATCC), Italy office, Sesto San Giovanni, Italy.

Eugenol, amphotericin B, calcein, sephadex G-50, LH-20, ergosterol (Erg-), pentamidine isethionate, propidium iodide (PI), and 1,6-diphenyl-1,3,5-hexatriene (DPH) were obtained from Sigma-Aldrich (Bornem, Belgium). Miltefosine was obtained from Santa Cruz Biotechnology (Heidelberg, Germany). calcein-AM included in a live/dead Viability/Cytotoxicity Kit for mammalian cells, Alamar blue and goat anti-Guinea Pig IgG Secondary Antibody, Alexa Fluor™ 488 were obtained from Thermo Fisher Scientific (Merelbeke, Belgium). The 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), l-α-phosphatidylinositol (PI—Liver, Bovine), and cholesterol (Chol—Ovine Wool) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). All organic solvents used were from VWR (Leuven, Belgium).