The pellet in UTC buffer was centrifuged for 10 min at 14,000 g and the complete supernatant was used for profiling on WCX ProteinChip® Arrays.
Extraction buffer was centrifuged for 15 min at 6 100 × g, the supernatant was filtered, and MgCl2 added to a final concentration of 50 mM in order to precipitate the microsomes [ 34].
Harvested cells suspended in Tris buffer were centrifuged at 17,000 g for 8 min at 4°C.
The cells suspended in the buffer were centrifuged at 10 000 g for 10 min at 4°C, and then the supernatant (containing the total protein fraction) was carefully transferred to a new tube.
For whole-cell proteins, approximately 50 mg mL−1 of cells or spheres (wet weight) was lysed by boiling in SDS sample buffer (Laemmli, 1970) for 10 min. For examination of released proteins, spheres that had been prepared by treatment with TL buffer were centrifuged at 10 000 g for 20 min. Supernatants were taken off and filtered through 0.22-μm sterile filters to remove any residual spheres.
To check the GEF-H1 expression level, transfected cells were rinsed with ice-cold PBS and suspended in cell lysis buffer B. Lysates were centrifuged for 15 min at 20,000 g, and supernatants were collected.
Briefly, 200 µl of buffer CER I supplemented with protease inhibitors were added to the homogenate and then sonicated on ice for 20 seconds and then incubated on ice for a further 10 min. The homogenate with 10 µl of CER II buffer was centrifuged at 12,000×g for 10 min at 4°C.
After 10 minutes, the red blood cells were lysed using 2 ml Pharm Lyse Buffer (BD Biosciences), and the samples were centrifuged for 5 minutes at 200×g at 20°C.
After a brief centrifugation, the Parafilm was removed, 150 μl of ice-cold lysis buffer was added, and the plates were centrifuged for 15 min at 490 × g at 4 °C.
