Bca reagent

Typically lots of 8–10 bottles, each containing700 ml cultures at ~3.5 – 4x10⁶ cells per ml were harvested by centrifugation at 2500 rpm using a GS3 rotor and washed with cold PBS yielding approximately 80 ml of packed cells. After the PBS wash, cells were resuspended with 10 times the cell pellet volume in ice cold lysis buffer (50 mMTris-HCl pH 7.4, 1 mM EDTA) containing 3X concentrated protease inhibitors: (Leupeptin (RPI) 3.0 μg/ml, Aprotinin (RPI) 6.0 μg/ml, Pefabloc (RPI) 150 μg/ml, Benzamidine (Fisher) 36 μg/ml, E64 (AG Scientific) 10.5 μg/ml. The resuspended cells were incubated on ice for 10 minutes then disrupted with 5 strokes of a 100 ml Dounce homogenizer. Sucrose buffer (25 ml of 1.5 M sucrose, 50 mM Tris pH 7.2) was added to the homogenate and mixed with 2 more strokes. Unbroken cells and cell debris were removed by centrifugation at 5000 rpm in a GS3 rotor. The crude membranes were collected by centrifugation of the GS3 supernatant in a SW28 rotor at 26,500 rpm for 30 minutes at 4°C. The membrane pellets were collected and resuspended in buffer A (see below) containing 2X protease inhibitors). Membranes were stored at −80°C prior to solubilization. Protein concentrations were measured using bicinchoninic acid assay (BCA reagent from Pierce Thermo Scientific).

Samples from the supernatants were diluted 1:30 v/v in deionized water and incubated with BCA reagent (ThermoPierce) for 30 min at 37°C. Absorbance of solutions at 560 nm was measured using a plate reader. The absorbance values were compared against a bovine serum albumin standard to determine protein concentrations.

A portion of the lung, liver, spleen, and the brain were used to prepare tissue homogenate by homogenizing them at approximately 20% w/v in 50 mM Tris-HCl (pH 7.4) buffer using a Dounce homogenizer on ice. A low-speed centrifugation was done to remove debris (4°C, 20 min, 1,000 × g) from the crude homogenates. All homogenates were stored at −80°C until analysis. Protein concentrations were determined using the BCA reagent (ThermoPierce) following the manufacturer’s instructions. Following completion of the reaction, all enzyme activities were normalized by the protein concentrations.

Whole-tissue homogenates (lung, liver, spleen, and brain) were prepared on ice at 20% w/v in 50 mM Tris-HCl (pH 7.4) buffer using a Dounce homogenizer. The crude homogenates were centrifuged at low speed to remove debris (4 °C, 20 min, 1,000 x g). For some studies, lung subcellular fractions were prepared: lungs were homogenized in sucrose buffer (50 mM Tris-HCl, 0.32 M sucrose, pH 7.4), centrifuged at low speed (4 °C, 5 min, 1,000 x g), and the resulting supernatant was centrifuged at high speed (4 °C, 60 min, 100,000 x g). The resulting pellet was washed and resuspended in sucrose buffer by sonication, then re-centrifuged (4 °C, 60 min, 100,000 x g); this wash step is critical for removing contaminating soluble blood components. The final pellet was sonicated in 400 μL of sucrose buffer to give washed lung microsomes. All homogenates and microsomes were stored at −80 °C until analysis. Protein concentrations were determined using the BCA reagent (ThermoPierce) with bovine serum albumin standards.