Pac5.1 v5 hisa

Drosophila Kc cells were cultured at Schneider's Drosophila medium (Sigma-Aldrich, USA) supplemented with 5% fetal bovine serum [HyClone, USA]32 (link). cDNAs of egfp, Mmp2, and Mmp2^GPI- were cloned into the pAc5.1/V5-HisA (Invitrogen) to obtain pAc5.1-egfp, pAc5.1-Mmp2, and pAc5.1-Mmp2^GPI-. Mmp1.f1, Mmp1.PC, and Mmp2 were cloned into the pUAST vector to obtain pUAST-Mmp1.f1, pUAST-Mmp1.PC, and pUAST-Mmp1.PC. The pUAST-DE-cadherin-GFP construct (Oda and Tsukita, 1999) was generously provided by Dr. Hiroki Oda. pActin-GAL4 plasmid was made by our lab. The constructs were transfected or co-transfected into Kc cells using Lipofectamine^TM2000 (Invitrogen, USA) as previously described32 (link). After 48 hours of incubation, the cells were collected by centrifugation, washed with PBS and homogenized in NP-40 lysis buffer (Beyotime). The MMP proteins in the cell lysate and/or culture medium were analyzed by Western blotting.

pAc5.1A-Luc (S2 cell expression vector) was created using the Luc gene from the
pGL3-Basic vector (Promega, Madison, WI, USA), which was ligated into the
EcoRI-NotI sites of pAc5.1/V5-HisA (Invitrogen). The Bowl 3’UTR (853 bp)
from Bowl cDNA (RE05342, Drosophila Genetic Resource Centre)
was ligated into the XhoI-XbaI sites of the pAc5.1A-Luc vector.

The helicase domain of Dcr-2 was cloned as a V5/3XFLAG tagged fusion protein into pAc 5.1 V5 His(A) (Invitrogen) using the restriction free cloning method. The 3XFLAG region from p3XFLAG-CMV-9 Expression Vector (Sigma-Aldrich) was amplified by PCR using primers Flag_F and Flag_R (Table S3, related to Figure 6). This PCR product was used as a primer for a second PCR using pAc 5.1 V5 His(A) as template. During this amplification step, the His tag was removed from the original plasmid. To clone the helicase of Drosophila melanogaster Dcr-2 in pAc 5.1 V5 3XFLAG, Dcr-2 helicase was amplified by PCR from cDNA from wild-type flies (w¹¹¹⁸) with primers Dicer-2_Flag_F and Dicer-2_Flag_R (Table S3, related to Figure 6). The purified PCR product was used as primer for a PCR using pAc 5.1 V5 3XFLAG as a template. The resulting plasmid, designated pAc5.1 Dcr-2 helicase V5 3XFLAG, carries the coding region of Dcr-2 helicase downstream of the Drosophila actin promoter and two tags for protein purification in the C-terminal region, V5 and 3XFLAG. Final plasmid was sequenced to verify the presence of inserts at the desired positions without mutations, deletions or insertions.