4.1. Plasmid Construction

To generate Myc-Usp10-PA and Flag-Ub expressing constructs, their corresponding coding sequences (CDS) were amplified from the cDNAs of Drosophila S2 cells and cloned into pAc-V5/HisB vector. Sequences encoding the N-terminal Myc epitope (MEQKLISEEDLNE) or Flag epitope (MDYKDDDDK) were added by PCR in place of the first Met codon of the respective CDS. Constructs of Myc-Usp10-PB, Myc-Usp10-PBN, and Myc-Usp10-PBC in pAc-V5/HisB vector were generated using Myc-Usp10-PA as the template. No tagged Usp10-PA was cloned into pUAST-attB vector by PCR for Usp10-PA expression in vivo. pAc-HA-Yki has been described previously [10]. All generating constructs in this study were confirmed by DNA sequencing.