Quick start bradford assay

Protein concentration of PSD preparations was determined using 1X Quickstart Bradford assay (BioRad). Thirty micrograms of PSD protein was prepared to contain 1X Novex NuPAGE LDS sample loading buffer (Invitrogen) with 100 mM DTT (BioRad), boiled for 10 min at 100˚C and loaded into 1 well of a 10-well Novex NuPAGE 3–12% Bis-Tris gradient gel (Invitrogen). Electrophoresis was performed under reducing conditions using the Novex NuPAGE SDS-PAGE system (Invitrogen) for 5 min. Gels were then stained with SimplyBlue SafeStain (Invitrogen) following the manufacturer’s instructions. Gel bands were excised and subjected to tryptic digestion using standard methods.

Animals were anesthetized by IP injection of 50 mg of Pentothal (Sigma, Kanagawa, Japan), lungs were lavaged twice with 1 mL ice cold physiological saline through a tracheal cannula, removed rapidly and immediately frozen in liquid nitrogen and stored at −80 °C for subsequent use. BALF from each sample was centrifuged (4 °C, 3000 rpm, 10 min). The supernatants were stored at −80 °C to determine the total protein concentration with Quick-Start Bradford assay (Bio-Rad, Marnesla-Coquette, France) and to assess metalloproteinases (MMPs) activity by zymography technique. Erythrocytes were removed from the cell pellet by hypotonic lysis followed by two washes with cold sterile saline. The cells were resuspended in 150 µL of physiological saline and an aliquot was used to evaluate total white cell count with a hemocytometer. For differential cellularity, the cell suspension was cytospun (Cytospin-2, Shandon Products Ltd., Levallois-Perret, France), fixed in methanol, and stained with Diff-Quick solution (Medion Diagnostics, Plaisir, France). Three hundred cells were counted with an oil immersion lens (1000×).

Target protein expression was determined by Western blotting as described elsewhere. Briefly, HepG2 cells were lysed in RIPA buffer, and extracted proteins were quantified using Quick Start Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were resolved on 12% polyacrylamide gels, transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), blocked and treated with primary Abs against TNF-α (Cell Signaling Technology; Cat#: 6945, Danvers, MA, USA), PPARα (Abcam; Cat#: ab3484, Cambridge, UK), or β-actin (Cell Signaling Technology; Cat#: 3700T, Danvers, MA, USA), followed by treatment with HRP-linked secondary Ab (Promega, Madison, WI, USA). Protein bands were developed using the SuperSignal West Femto ECL kit (Thermo Scientific, Waltham, MA, USA), and images were captured (ChemiDoc MP imaging system, Bio-Rad, Hercules, CA, USA).