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Fig. 2. Ubiquitylation of Pma1-10 at or before arrival at the plasma membrane is reversible. Induction and shut-off of synthesis of HA-tagged Pma1 was at 37°C. (A) Western blot with anti-HA antibody before HA-Pma1-10 synthesis (0), after induction for 1 hour (on) and chase for 2 hours (off) in end3-1 (KKY81) and END3 (KKY80) cells. (B) HA-tagged mutant was immunoprecipitated with anti-HA from end3-1 and END3 cells; wild-type HA-Pma1 was immunoprecipitated from END3 cells. Immunoprecipitates were blotted with anti-ubiquitin. Arrow indicates position of Pma1 protein. After stripping, the filters were blotted again with anti-HA.