Fig. 2

Ubiquitination and degradation of OsSKIPa by two RING-type E3 ligases. a Confirmation of the interactions between OsSKIPa and 11 E3 ligases in yeast. Yeast strain AH109 containing BD-OsSKIPa was mated with yeast strain Y187 containing the E3 ubiquitin ligase library. OsRING77 (indicated in blue) was identified in a previous study. DDO, SD-Leu-Trp, QDO, SD-Leu-Trp-His-Ade. b Three types of E3 interactors with OsSKIPa. The pink color line represents the identified interaction using the UbE3 library, and the blue color line represents the literature-reported interaction. c Co-IP assay of OsSKIPa with eight candidate E3 ligases in planta. N. benthamiana leaves were agro-infiltrated with the indicated plasmid combinations. Proteins were extracted and immunoprecipitated with anti-GFP/Agarose beads. Immunoblot detection was performed using anti-GFP or anti-HA antibody as indicated. d Degradation assay of OsSKIPa by three E3 ligases (OsRING77, OsRING113, and OsPUB46) with or without 50 μM MG132. N. benthamiana leaves were agro-infiltrated with plasmids harboring OsSKIPa-GFP and three candidate E3 ligases fused with HA tag. GUS-HA was used as a negative control. The protein abundance of OsSKIPa and the candidate E3 ligases was detected by immunoblotting using anti-GFP or anti-HA antibody, respectively. ACTIN was used as an internal control for protein loading. OsSKIPa transcript level was measured by RT-PCR, and ACTIN was used as the internal control. e Ubiquitination assay of OsSKIPa by OsRING77 in vitro. GST-OsSKIPa protein purified from E. coli was incubated with MBP-OsRING77 and E1, E2, Ub, and ATP in the reactions. Immunoblot analysis was performed with anti-ubiquitin, anti-MBP, or anti-GST antibody. f Ubiquitination assay of OsSKIPa by OsRING113 in vitro. GST-OsSKIPa protein purified from E. coli was incubated with MBP-OsRING113 and E1, E2, Ub, and ATP in the reactions. Immunoblot analysis was performed with anti-ubiquitin, anti-MBP, or anti-GST antibody.