Pad adenoviral vector

For the knockdowns, Rab27a and Rab27b, as well as Rab31 microRNA (miRNA)–expressing vectors, were constructed by inserting specific nucleotide sequences into pcDNA6.2-GW/EmGFP-miR plasmid harboring a Pol II promoter obtained from Thermo Scientific. These sequences are fused with GFP coding sequence. The synthesized oligonucleotides were annealed and ligated into pcDNA6.2-GW/EmGFP-miR according to the manufacturers’ instructions. For Rab27a and Rab27b, tandem miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: Rab27a miRNA1, 5′-AAACTTTGCTCATTTGTCAGG-3′; Rab27a miRNA2, 5′-TTAACTGATCCGTAGAGGCAT-3′; Rab27b miRNA1, 5′-ATTGACTTCCCTCTGATCTGG-3′; and Rab27b miRNA2, 5′-TTTCCCTGAAGATCCATTCGG-3′. For Rab31, miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology. Sequences used were as follows: miRNA1, 5′-TTTCTTTGCAGGAAACGTCCC-3′ and miRNA2, 5′-TAAACTGAAGGCCATGTTGCG-3′. Viral particles were produced, and cells were infected as described before (51 (link)).

For Rab27 Knock-down, Rab27a and Rab27b micro-RNA (miRNA)-expressing vectors were constructed by inserting an inverted repeat of Rab27-specific 21-nucleotide sequences into pcDNA6.2-GW/EmGFP-miR plasmid harboring PolII promoter obtained from Thermo Scientific. These sequences are fused with GFP coding sequence. For miRNA targeting of human Rab27a and Rab27b, the synthesized oligonucleotides were annealed and ligated into pcDNA6.2-GW/EmGFP-miR according to manufacturers’ instructions. The oligonucleotides used are in Table 1 (underline indicates RNA interference target sequences). All plasmids were verified by DNA sequencing and tested for the Knock-down (unpublished data). The four miRNA were cloned in tandem into one pcDNA6.2-GW/EmGFP-miR plasmid according to manufacturers’ instructions. Tandem miRNA sequences were transferred into pAd adenoviral vector from Thermo Scientific using Gateway technology.
For shRNA targeting of human STUB1 and miRNA targeting of human Rab27a/b, viral particles were produced and cells were infected as described before [35 (link)].