Sodium dodecyl sulfate (sds)

Prior to electrophoretic analysis, MVs and the culture supernatants were diluted with an equal volume of native-PAGE or SDS-PAGE buffer (0.06 M of Tris-HCl (Amersham Pharmacia Biotech, Buckinghamshire, UK), pH 6.8, 20% glycerol (Wako Pure Chemical Industries Ltd., Osaka, Japan), 0% or 1% (wt/vol) SDS (Wako), 0% or 1% 2-mercaptoethanol (2-ME, Merck kGaA), and 0.0012% bromophenol blue (Wako)). The SDS-PAGE samples were heated at 100 °C for 5 min just prior to loading on the gel. The native-PAGE or SDS-PAGE samples were then ran on a 12.5% polyacrylamide gel (e-PAGEL, ATTO Corp., Tokyo, Japan) in 0.025 M of Tris-HCl, 192 mM of glycine (Wako) and 0% or 0.1% (wt/vol) SDS. Electrophoretic separation of the proteins was carried out for 70 min at 25 mA. Gels were stained with Coomassie Brilliant Blue (CBB) and ethidium bromide to observe the proteins and DNA, respectively.

Stool samples collected from mice were immediately frozen using liquid nitrogen and stored at −80 °C until use. Bacterial genomic DNA was isolated as described previously with modifications51 (link)52 (link). The bacterial pellet was suspended and incubated with lysozyme (15 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 1 h in 100 mM Tris-HCl/10 mM EDTA (10 × TE). Purified achromopeptidase (Wako Pure Chemical Industries, Ltd) was added at a final concentration of 2000 U/ml and then incubated at 37 °C for 30 min. The suspension was treated with 1% (wt/vol) sodium dodecyl sulfate (Wako Pure Chemical Industries, Ltd) and proteinase K (1 mg/ml, Merck Japan, Tokyo, Japan) and incubated at 55 °C for 1 h. The lysate was treated with phenol/chloroform/isoamyl alcohol, and DNA in the aqueous phase was precipitated by the addition of ethanol and pelleted via centrifugation at 5,000 × g at 4 °C for 15 min. The DNA pellet was rinsed with 75% ethanol, dried, and dissolved in 1 × TE. DNA samples were purified by treatment with RNase A (1 mg/ml, Wako Pure Chemical Industries, Ltd) at 37 °C for 30 min and precipitated by the addition of equal volumes of a 10% polyethylene glycol solution (PEG6000-2.5 M NaCl). DNA was pelleted via centrifugation at 18,000 × g at 4 °C for 10 min, rinsed with 75% ethanol, and dissolved in 1 × TE.

Rhodamine 123, l‐NAME, and sodium dodecyl sulfate (SDS) were purchased from Wako (Tokyo, Japan). ISOGEN was purchased from Nippongene (Tokyo, Japan). DMEM, RIPA buffer, protease inhibitor cocktail, and β‐Actin antibody were purchased from Sigma (MO, USA). Fetal bovine serum (FBS) and house serum (HS) were purchased from Gibco (NY, USA). MTT or 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide and l‐NAME were purchased from Dojindo (Kumamoto, Japan). l‐citrulline was supplied by Kyowa Hakko Bio Co., Ltd., Tokyo, Japan) while PGC‐1α (3G6) antibody was purchased from Cell Signaling Technology (Hertfordshire, UK). Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (6C5) was purchased from Santa Cruz Biotechnology (CA, USA).

HA with a mean molecular weight of 30,000 (polydispersity index of 1.212) was purchased from MRC polysaccharide Corp., Ltd (Toyama, Japan). RPMI 1640 medium, 4-dimethylaminopyridine (DMAP) isoflurane, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Tween 20, sodium dodecyl sulfate (SDS) and zinc acetate were purchased from Wako pure chemical (Osaka, Japan). Protoporphyrin IX (PP) and cetyltrimethylammonium bromide (CTA) were from Sigma-Aldrich Chemical (St. Louis, MO, USA). Water-soluble carbodiimide (WSC) was purchased from Dojindo Laboratory, Kumamoto, Japan. Additionally, 2,2,6,6-Tetramethyl-4-piperidone (4-oxo-TEMP) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Esterase from porcine liver was purchased from Roche Diagnostics GmbH (Mannheim, Germany). The other reagents and solvents of a reagent grade were from commercial sources and were used without further purification.