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| PCR Cloning and Subcloning | Mouse anti-HA was purchased from Youke Biotechnology (Shanghai, China), mouse anti-Myc and anti-GST, and rabbit anti-S tag antibodies were from GenScript (Nanjing, China). RACK1B was amplified from cDNA by PCR and cloned into pMD18 with HA tag fused at N-terminus. RACK1B sequences with the following mutations, K50R, K272R, K276R, K281R, K291R, 3KR-K50, 3KR-K276, 3KR-K281, 3KR-K291 and 4KR, were synthesized (GenScript) and cloned into pUC57 with HA tag fused at N-terminus.To verify the interaction of RACK1B with SCE1A and SIZ1, coding sequences of RACK1B, SCE1A and SIZ1 were cloned into pGS-21a (GenScript) to generate GST-RACK1B, GST-SCE1 and GST-SIZ1, respectively.Cultures were then induced with 0.1 mM IPTG for 2 h at 25 °C. Cells were harvested in binding buffer (50 mM Tris-HCl pH 7.8, 200 mM NaCl, 1% Triton X-100, and 10% glycerol) at a ratio of 1:10. For the SIZ1-RACK1B interaction, 500 μ L crude extract of GST-SIZ1 for each individual trial was incubated with 30 μ L glutathione-Sepharose beads (GenScript) for 1 h on ice. | Get A Quote |
The highly conserved eukaryotic WD40 repeat protein， Receptor for Activated C Kinase 1 (RACK1)， is involved in the abscisic acid (ABA) response in Arabidopsis. However， the regulation of RACK1 and the proteins with which it interacts are poorly understood. Here， we show that RACK1B is sumoylated at four residues， Lys50， Lys276， Lys281 and Lys291. Sumoylation increases RACK1B stability and its tolerance to ubiquitination-mediated degradation in ABA response. As a result， sumoylation leads to enhanced interaction between RACK1B and RAP2.6， an AP2/ERF family transcription factor. RACK1B binds directly to the AP2 domain of RAP2.6， which alters the affinity of RAP2.6 for CE1 and GCC cis-acting re... More
