Figure 4.

Plasmid‐encoded β‐est AID and the effect of TIR1 protein localization on targeted depletion efficiency. (a) Plasmid map of pZTRL. (b) Western blot of TIR1 and AID‐target Prp22‐AID*‐6FLAG in three different strains preincubated with β‐estradiol for 30 min (min) prior to auxin addition. Equal amounts of total protein were loaded in each lane, and Pgk1 is included as a visual loading control. In the first strain (lanes 1–5), both Z4EVpr‐OsTIR1‐V5 and Pact1‐Z4EV expression cassettes are located in pZTRL centromeric plasmid; whereas in strains PZ4EV‐TIR1 (lanes 6–10) and PZ4EV‐NTIR1 (lanes 11–15), they are genomically encoded. In PZ4EV‐NTIR1, an SV40 nuclear localization signal was included in the N‐terminal of TIR1. (c) Quantification of western blot signal shown in panel B. Error bars represent sd of two biological replicates. Only one representative blot is shown