Figure 4.

Untargeted Ubiquitin Machinery by Secdin.

(A) Total protein ubiquitination pattern of wild-type (Col-0) seedlings treated with Secdin (50 µM) at different times and subjected to protein gel blot (Western blot [WB]) analysis with an α-Ub P4D1 antibody. Tubulin (TUB) (α-TUB antibody) was used as a loading control.

(B) Total microsomal protein ubiquitination pattern of DMSO- or Secdin-treated BRI1-mCitrine (mCit)-expressing seedlings detected with an α-Ub P4D1 antibody. Tubulin was used as a loading control.

(C) Protein gel blot analysis of the ubiquitination pattern of immunoprecipitated (IP) BRI1-mCit and BRI1_25K-R-mCit after microsomal protein isolation. Detection with an α-GFP antibody recognizing the mCit tag was done to estimate the BRI1-mCit protein loading. All chemical treatments ([B] and [C]) were done for 5 h in the presence of MG132 (50 µM) to enrich for ubiquitinated proteins.

(D) Unaffected AMSH3 DUB activity by Secdin. Fluorescence-based DUB assay with diubiquitin TAMRA (diUb-TAMRA) was performed with (red) or without (blue) preincubation of AMSH3 with Secdin (100 µM). DiUb-TAMRA alone was used as a negative control (black).

(E) Protein gel blot detection of AMSH3-DUB activity with K63-linked polyubiquitin (Ub_2-7) chains. For the DUB assay, AMSH3 was incubated with the polyubiquitin chains with or without 10-min preincubations with 50 µM Secdin. The assay was terminated at the indicated time points and the reaction mixture was subjected to protein gel blot analysis with an α-Ub P4D1 antibody.