Flag tagged ubiquitin

The in vitro ubiquitination assay was performed as described previously29 (link). In brief, PUB12, PUB13, PYR1 and UBC8 (E2) were separately cloned into the pGEX-4T-1 vector, ABI1 was fused into the pET-28a vector. The recombinant proteins were extracted from E. coli strain BL21 (DE3). The primers used for this assay are listed in Supplementary Table 1. The fusion proteins were purified with glutathione-sepharose and Ni-sepharose. A 250-ng quantity of wheat (Triticum aestivum) E1, 500 ng of purified E2-GST (UBC8), 1.25 μg of Flag-tagged ubiquitin (Boston Biochem, cat. no. U-120), 1 μg of purified PUB12/13-GST, 500 ng of ABI1-His substrate, 500 ng PYR1-GST and 5 μM ABA were added to 30 μl of ubiquitination reaction buffer (50 mM Tris-Cl pH 7.5, 2 mM ATP, 5 mM MgCl₂, 2 mM DTT)29 (link). After 2 h at 30 °C with oscillation in a thermomixer (Eppendorf), the reactions were stopped by adding 4 × SDS loading buffer; the samples were the boiled at 100 °C for 5 min. The products were electrophoresed on a 10% SDS–polyacrylamide gel electrophoresis (PAGE) gel and detected with anti-His and anti-Flag antibody by western blotting.

The CERK1-HA, AtUBC8 and PUB12 were subcloned into a pCold GST vector (Takara). The recombinant proteins were expressed using a Cold-Shock bacterial expression system (Takara), and purified using Glutathione Sepharose 4B (GE Healthcare). The CERK1-HA and AtUBC8 proteins were purified by on columm Turbo3C Protease (Accelagen) digestion. The in vitro ubiquitination assays were performed as described with some modifications [17 (link)]. The reactions contained 1μg of purified CERK1-HA, 500 ng of His6-E1(human UBE1) (Boston Biochem), 1μg of purified E2 (AtUBC8), 2.5 μg of FLAG tagged ubiquitin (Boston Biochem) and 4μg of purified GST-PUB12 in the ubiquitination buffer (0.1 M Tris-HCl pH 7.5, 25 mM MgCl2, 2.5 mM dithiothreitol, 10 mM ATP) to a final volume of 60 μl. The reactions were incubated at 30°C for 2 hr, and then stopped by adding SDS sample buffer and boiled at 100°C for 5 min. The samples were then separated by SDS–PAGE and the ubiquitinated substrates were detected by Western blotting analysis using an anti-FLAG antibody (F3165; Sigma-Aldrich) or an anti-HA antibody (11867423001; Roche).

For the in vitro ubiquitination assay Flag-tagged ubiquitin (25 µg), E1 (150 ng), UbcH5c (300 ng) or UbcH5a (300 ng) (Boston Biochem) and freshly immunoprecipitated proteins (PP2A and NOSIP 1 µg each) from MEFs were incubated with 2 mM ATP for 1 h at 37°C in ubiquitin assay buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl₂). To stop the reaction, SDS-loading buffer was added and the samples were boiled at 95°C for 1 minute. The samples were subsequently analysed by SDS-PAGE followed by immunoblotting.
For the in vivo ubiquitination assay pBabe-puro empty vector, pBabe-Flag-ubiquitin and/or pBabe-NOSIP were transfected into Phoenix-ECO cells (ATCC) using Gene Juice (Novagen). After 48 hours of transfection, released retroviruses were filtered and used for infection of MEFs using polybrene (4 µg/ml). 48 hours post infection MEFs were used for immunoprecipitation. For proteasome inhibition cells were treated with 20µM MG-132 for 5 h before immunoprecipitation.