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. 2015 Feb 6;11(2):e1004629. doi: 10.1371/journal.ppat.1004629

Figure 2. Ubiquitination of SPIN6 in vitro and degradation of SPIN6 in N. benthamiana by SPL11.

Figure 2

(A) In vitro ubiquitination of SPIN6 by SPL11. The ubiquitination of GST:SPIN6 by MBP:SPL11 and the E3 ligase activity of SPL11 were detected by immunoblot with the anti-GST antibody and anti-ubiquitin antibody, respectively, in the presence of Arabidopsis E1 (At5g06460), E2 (AtUBC10, At5g53300) and ubiquitin. MBP and mutated SPL11 without E3 ligase activity (SPL11m) fused with MBP were used as the negative controls. Immunoblot with anti-MBP antibody was conducted as a loading control. (B) Degradation of SPIN6 promoted by SPL11 in N. benthamiana. Either SPL11:Myc or SPL11m:Myc was co-expressed with GFP:SPIN6 by agro-infiltration in N. benthamiana. Tissues were harvested at 3 days after the infiltration. MG132 (50μM), an inhibitor of 26S proteasome system, was infiltrated at 18 h before sampling with DMSO (-) as a control. RFP was expressed as an internal control.