Plant Cell (2014) 26: 712-728 doi: 10.1105/tpc.113.122234
The authors were made aware of mistakes in the preparation of Figures 6B, 6C and 6D. Figure 6B shows immunoblots corresponding to co-immunoprecipitation assays between 3HA-PYL8 and DDA1-GFP fusion proteins transiently expressed in Nicotiana benthamiana epidermal leaf cells in the presence and absence of ABA. The lower panel shows RPT5 protein bands, used as loading control, that were inadvertently taken from the RPT5 blot shown in Figure 5G since both figures were prepared at the same time. To dispel any doubt, the co-immunoprecipitation assays were repeated and a corrected version of Figure 6B was prepared. Figure 5G stays the same as it is correct.
Figure 6.
(corrected). ABA reduces PYL8 polyubiquitination but not its interaction with DDA1
(A) ABA does not disrupt PYL8 and DDA1 interaction in yeast two hybrid assays. Yeast transformed with the indicated constructs were grown on selective plates with 100 mM ABA and compared to mock controls. Selective media contained different concentrations of 3-amino-1,2,4-triazole (3AT; ranging 0.5-2 mM). pGAD-T and pGBK-53 clones (Clontech) were used as the positive control (Control +).
(B) DDA1-GFP associates to 3HA-PYL8 in the presence of ABA in vivo. 3HA-PYL8 and DDA1-GFP fusions were transiently expressed in 3-week-old Nicotiana benthamiana leaves treated with MG132 in the presence or absence of 50 μM of ABA. Total extracts (INPUT) and immunoprecipitates (IP) were subjected to immunoblot analysis with anti-GFP and anti-HA antibodies. Anti-RPT5 antibody was used for loading control purposes (also in [C and D]).
(C) Immunoblot showing DDA1-GFP stabilization by proteasome inhibitor MG132. Nine-day-old DDA1-GFP seedlings were treated or not for 2 h with 50 μM MG132. DDA1-GFP was detected using anti-GFP. Wild-type plant (WT) extracts were used as immunoblot negative control.
(D) Time course of relative abundance of DDA1-GFP in 9-d-old seedlings treated with 50 μM cycloheximide (CHX) in the presence or absence of 50 μM ABA.
(E) ABA treatments reduce 3HA-PYL8 polyubiquitination. For affinity purification of polyubiquitinated 3HA-PYL8, total protein extracts obtained from oe3HA-PYL8 seedlings treated or not with ABA were incubated with Ub-binding p62 resin or with empty agarose resin (negative control). Anti-Ub was used to detect total ubiquitinated proteins. Anti-HA antibody allowed detection of 3HA-PYL8 and its ubiquitinated forms (Ub(n)-3HA-PYL8; shown by brackets). The arrow indicates the position of a non-specific protein detected by anti-HA.
Figures 6C and 6D show the effect of proteasome inhibitor MG132, and cycloheximide and ABA, respectively, in the accumulation of DDA1-GFP protein; the lower panels correspond to loading controls as shown by Ponceau staining of the membrane. While preparing the lower panels some bands were selected twice by mistake. To eliminate any concerns, both assays were repeated and corrected versions of Figures 6C and 6D were prepared. For more accurate loading controls, anti-RPT5 immunoblots were used instead of Ponceau staining of the membranes. The corrected version of Figure 6, with new panels B, C, and D is shown below. These corrections do not alter any of the findings or conclusions presented in the manuscript. We apologize for any inconvenience caused by these mistakes.
Editors’ note: This correction notice was reviewed by members of The Plant Cell editorial board. The authors are responsible for providing a complete listing and accurate explanations for all known errors or instances of inappropriate data handling or image manipulation associated with the original publication.