Pcmv 3tag 1a

The ESRP1, hMENA11a and hMENAΔ11a were cloned in pCMV-3Tag1a (Agilent, 240195) overexpression plasmid from MCF7 cDNA using DreamTaq DNA Polymerase (Thermo Fisher Scientific, EP0702). Primers were as follows: ESRP1 Fw (5′-CGGGATCCATGACGGCCTCTCCGGATTA-3′), ESRP1 Rev (5′-CCCAAGCTTTTAAATACAAACCCATTCTTTGGG-3′), hMENA11a/hMENAΔ11a Fw (5′-CGGAATTCATGAGTGAACAGAGTATCTGTCAGG-3′) and hMENA11a/hMENA11Δa Rev (5′-ACGCGTCGACCTATGCAGTATTTGACTTGCTCAGTT-3′). ESRP1 was cloned between the BamHI F and HindIII R sites and hMENA11a/hMENA11Δa was cloned between the EcoRI F and Sa1I R sites.

Plasmids expressing FLAG-tagged hRpn13 or hRpn13^1–279 were generated commercially (GenScript) by inserting synthesized coding sequence for full-length hRpn13 (NM_007002.3) or for residues 1–279 between the BamHI and HindIII restriction sites of pCMV-3Tag-1a (Agilent Technologies, 240295). Unmodified pCMV-3Tag-1a was used as empty vector (EV) control.

pcDNA3.1- ERα and 2XERE-Tk-LUC vectors have been previously described (19 (link), 20 (link)). Human full-length IFI27/ISG12 mRNA (GenBank TM accession no. NM_001130080) was amplified by RT-PCR and cloned into the mammalian expression vector pCMV-3Tag-1A (Agilent Technologies, Santa Clara, CA). The resulting vector is referred as pCMV-3Tag-ISG12.

ESRP1- pCMV-3Tag-1A clone was generated by PCR-amplified full-length ESRP1 fragment using cDNA derived from MCF7 cells as a template. This PCR-amplified full-length ESRP1 fragment was cloned into pCMV-3Tag1a (Agilent, 240195) vector between BamHI F and HindIII R restriction sites. E2F1 and RB1 expression plasmids were a kind gift by Dr. Sudhakar Baluchamy, Department of Biotechnology, Pondicherry University, INDIA. The details of the primers are listed in Supplementary Table S3.
To generate promoter constructs, human ESRP1 (Gene ID: 54845) promoter sequence was retrieved from the Eukaryotic Promoter Database (EPD) (https://epd.vital-it.ch/). Different lengths of ESRP1 promoter constructs were PCR amplified from genomic DNA derived from MCF7 cells and inserted/ligated into pGL3 basic vector (Promega) between KpnI and NheI restriction sites. The primers used in promoter constructs are listed in Supplementary Table S4.
The site-directed mutant construct of the ESRP1 promoter was prepared using oligonucleotides harboring mutations in the E2F1 binding site. The wild type ESRP1 −472 promoters was used as a template. The ESRP1 promoter SDM was confirmed by DNA sequencing after the digestion of non mutated vector with the endonuclease DpnI (TaKaRa, 1235 A). The details of the oligonucleotides are listed in Supplementary Table S5.