Pcdna3 ha rbx1

The open reading frame of mouse SPOP (Uniport: Q6ZWS8-1) and its corresponding mutants were cloned into pcDNA4/TO/myc-His vector (Thermo Fisher) to produce C-terminally Myc-tagged proteins. pcDNA3-Myc-Cullin3, and pcDNA3-HA-Rbx1 were obtained from Addgene (#19893 and #19897, respectively).^{34 (link)} Ub-His and BRD3-Flag plasmids were kind gifts from Wenyi Wei (Harvard Medical School) and Joel Mackay (University of Sydney), respectively. The full-length human SPOP gene encoding residues 1-374 (His-SUMO-SPOP^1-374) was cloned into a pEXP vector containing an N-terminal hexa-histidine-SUMO tag for bacterial expression and affinity purification. Rolling circle mutagenesis of this vector backbone was used to create all SPOP point mutants used in this study. A synthetic gene for BRD3, codon-optimized for E. coli, was ordered from Genscript in a pET28a vector. A hexa-histidine tag followed by a TEV protease cleavage site was placed at the N-terminus of the open reading frame.