反应详情

Experiments were performed following the methods previously described by Dr. Stamler's laboratory (39). Cell lysates containing Flag or Myc-tagged MKP7 protein were treated with the indicated concentrations of GSNO followed by the addition of 2 volumes of HEN buffer and freshly prepared S-methyl methanethiosulfonate (10% v/v in N,N-dimethylformamide) and SDS (25% v/v) to final concentrations of 0.1 and 2.5%, respectively. Following frequent vortexing at 50° C. for 1 hour, proteins were precipitated with 3 volumes of acetone at −20° C. for 30 min. The proteins were recovered by centrifugation at 5,000×g for 5 min, followed by gentle rinsing of the pellet with 3×1 ml acetone. The pellets were then resuspended in 240 μl of HEN buffer containing 1% SDS. The resuspended protein was precipitated, rinsed and resuspended once more. For labeling, the blocked samples were mixed with 0.1 volume of biotin-HPDP (2.5 mg/ml in Me2SO) and 0.1 volume of HEN (control) or freshly prepared sodium Ascorbic acid in HEN buffer. Labeling reactions were performed in the dark at room temperature for 1 h unless otherwise indicated. To detect an MKP7 SNO protein from cells or lysates, the labeling reaction was acetone-precipitated as previously described. The washed pellet was resuspended in 250 μl of HEN/10 (HEN diluted 10-fold into H2O) containing 1% SDS, followed by addition of 750 μl of neutralization buffer (25 mM HEPES, 100 mM NaCl, 1 mM ETDA, 1% Triton X-100, pH 7.5). This material was incubated overnight at 4° C. with 40 μl of streptavidin-agarose beads. The beads were washed with 3×1 ml of wash buffer (neutralization buffer+500 mM NaCl) and 2×1 ml of neutralization buffer. The dried beads were eluted with 50 μl of HEN/10+1%-mercaptoethanol. The eluted mixture was then analyzed by SDS-PAGE, followed by immunoblotting with anti-Flag-HRP (1:5000) or antibodies.

1. additionCell lysates containing Flag
2. additionfollowed by the addition of 2 volumes of HEN buffer
3. customat 50° C.
4. customfor 1 hour
5. customproteins were precipitated with 3 volumes of acetone at −20° C. for 30 min
6. customThe proteins were recovered by centrifugation at 5,000×g for 5 min
7. washby gentle rinsing of the pellet with 3×1 ml acetone
8. additionbuffer containing 1% SDS
9. customThe resuspended protein was precipitated
10. washrinsed
11. additionFor labeling, the blocked samples were mixed with 0.1 volume of biotin-HPDP (2.5 mg/ml in Me2SO) and 0.1 volume of HEN (control) or freshly prepared sodium Ascorbic acid in HEN buffer
12. customdark at room temperature
13. customfor 1 h
14. customthe labeling reaction
15. customprecipitated
16. addition10 (HEN diluted 10-fold into H2O)
17. additioncontaining 1% SDS
18. additionfollowed by addition of 750 μl of neutralization buffer (25 mM HEPES, 100 mM NaCl, 1 mM ETDA, 1% Triton X-100, pH 7.5)
19. washThe beads were washed with 3×1 ml of wash buffer (neutralization buffer+500 mM NaCl) and 2×1 ml of neutralization buffer
20. washThe dried beads were eluted with 50 μl of HEN/10+1%-mercaptoethanol