Figure 2.

The auxin IAA induces rapid depletion of mAID-tagged GRASP55 and 65. (A) Homozygous RC55, RC65, and RC65+55 cells inducibly expressing TIR1-2xMyc were treated with doxycycline for 6 h (short-term dox) and then with the auxin IAA for a further 2 h to degrade GRASP55 and/or 65. Cell lysates were immunoblotted with the indicated antibodies. (B) RC55, RC65, and RC65+55 cells were treated with doxycycline for 6 h and with IAA for a further 2 h as in A. Cells were then immunolabeled for GRASP55 (red) and GRASP65 (green) and stained for DNA (blue). Scale bar, 10 µm. (C) Quantitation of the fluorescence signal of GRASP55 and 65 in the Golgi region from B for RC55, RC65, and (S1B) for RC65+55. n = 3 independent experiments with >50 cells analyzed per experiment and condition. ** P < 0.01; *** P < 0.001; ns, not significant. Error bars represent mean ± SD. (D and E) GRASP55 does not compensate for the loss of GRASP65 to stabilize GM130. RC65 cells were treated with doxycycline for 6 h before adding IAA for 2 d (long-term IAA) to deplete GRASP65. GM130 (D) and GRASP55 (E) were then immunoprecipitated from the cell lysates and analyzed by Western blotting. * denotes unspecific band. (F and G) Simultaneous degradation of GRASP55 and 65 causes a delayed reduction of GM130 levels. RC65+55 cells expressing TIR1 for 6 h were treated with IAA. Cell lysates were collected at the indicated time points and subjected to Western blotting with the indicated antibodies. * denotes unspecific band. The intensities of each band are shown in G.