FIGURE 7:

S54 and S334 regulate TIS11b protein stability. (A) COS7 cells were cotransfected with 10 ng of pTarget plasmids encoding WT TIS11b (WT), TIS11b S54A, TIS11b S54D, TIS11b S334A, or TIS11b S334D mutants and then treated with cycloheximide (CHX) as described in Materials and Methods. The half-life of WT or mutant TIS11b was analyzed by Western blot. All the membranes were exposed simultaneously for 15 s. Quantification of TIS11b steady-state levels (TIS11b/actin ratio) at t = 0 yielded the ratios 1, 0.61, 1.27, 0.85, and 1.58 for WT TIS11b, TIS11b-S54A, S54D, TIS11b-S334A, and TIS11b-S334D mutants, respectively. (B) TIS11b protein levels were normalized to actin levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. (C) Calculated half-lives of TIS11b from four independent experiments. Half-lives of TIS11b mutants were compared with the half-life of WT TIS11b. p Values and 95% confidence intervals (CIs) were determined using an F-test.