Figure 4.

Knockdown of PAF49 results in the rapid degradation of PAF53, but not vice versa.A, cells were treated with 1 mM IAA or vehicle for 3 h to knock down AID-PAF49 levels. Cells were then harvested and lysed in HEPES lysis buffer. The expression of AID-PAF49 and PAF53 was determined by Western blot analysis. B, three independent repeats of the Western blot experiment presented in A were performed. The levels of PAF53 were corrected for β-actin. The data were analyzed by a two-tail t test, p < 0.0001. C, cells with PAF53-AID were treated with 1 mM IAA or vehicle for 3 h and were harvested and lysed in HEPES lysis buffer. The expression of PAF53-AID and PAF49 was determined via Western blot analysis. D, three independent repeats of the Western blot experiment presented in C were performed. The levels of PAF49 were corrected with β-actin. A two-tail t test was performed to test for significance, p = 0.3007. E, cells expressing AID- PAF49 were treated with 1 mM IAA for 3 h and were harvested and lysed at the indicated time points: 0, 15, 30, 60, 120, and 180 min after IAA treatment. Western blot analysis was performed to determine the protein levels of AID-PAF49 and PAF53 at each time point. F, three independent repeats of the experiment presented in E were performed. The levels of AID-PAF49 and PAF53 were corrected for the β-actin content of the sample. The change in AID-PAF49 and PAF53 levels with time were plotted with a nonlinear fit with GraphPad Prism. The dotted lines indicate the approximate half-life of each protein in these experiments.