FIG. 6.

Dephosphorylation rate of phospho-SLN1-R1. (A) Autoradiograph showing dephosphorylation of phospho-SLN1-R1 as a function of time. Phospho-YPD1 (0.5 μM) was incubated with a 20-fold molar excess of SLN1-R1 in 50 mM Tris-HCl (pH 7.5)–5 mM MgCl₂–1 mM DTT at room temperature for 1 min to allow complete transfer of the phosphoryl group. An aliquot was then removed, mixed with stop buffer, and labeled t₀. Subsequently, aliquots were removed at specific time points over the course of 80 min and mixed with stop buffer to terminate the reaction. Proteins were separated on SDS–15% polyacrylamide gels and analyzed as indicated in the legend to Fig. 3. (B) The autoradiographs were further analyzed by scanning laser densitometry to determine the fraction of phospho-SLN1-R1 remaining. Dephosphorylation of phospho-SLN1-R1 followed first-order rate kinetics, and the rate constant and half-life of phospho-SLN1-R1 were determined accordingly. The lines represent computer-generated least-squares fitting to a single exponential or a linear relationship (inset). Data shown are from a representative experiment which was performed multiple times.