Fractionation

1. All steps subsequent to the incubation of the cells with or without treatment are performed at 0-4°C.

2. Wash cells (four culture dishes [diameter 10 cm]/group) three times with 10 mL of HES buffer.

3. Scrape cells vigorously with a rubber disposable cell scraper in HES buffer (8 mL; 2 mL per dish) containing 1 mM PMSF, 10 ^g/mL pepstatin, 10 ^g/mL aprotinin, and 5 ^g/mL leupeptin and homogenize by passing the cells through a 22-gauge needle 10 times (see Note 7).

4. Centrifuge homogenate at 19,000g in a fixed-angle rotor (e.g., JA20 rotor) for 20 min (see Fig. 1).

5. Remove the supernatant and centrifuge for 20 min at 41,000g (JA20 rotor). The yielding pellet is designated as the HDM fraction (see Note 8).

6. Centrifuge the supernatant for 75 min at 180,000g to pellet the LDM fraction.

7. The supernatant of the 180,000g spin contains the cytosol, and this CYT fraction can be concentrated by centrifugation in Centricon tubes.

8. The pellet from the first centrifugation (19,000g, step 4) is resuspended in 5 mL of HES buffer and centrifuged again at 19,000g for 20 min. Resuspend the pellet again in 5 mL of HES buffer, and layer onto a 6.3-mL sucrose cushion (38.5%) and centrifuge for 60 min at 100,000g in a swing-out rotor (e.g., SW41, Beckman).

9. The resulting pellet contains nuclei and mitochondria and is brownish in color. The PM fraction is collected from the top of the sucrose cushion (white fluffy band at sucrose cushion interface), resuspended in 10 mL HES, and repelleted by centrifugation at 40,000g for 20 min.

10. Resuspend all pellets in 0.25-1.0 mL HES to a final protein concentration of 1-5 mg/mL and store at -20°C (see Notes 9 and 10).

11. Fractions are then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis (see Note 11).

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