Pcnda3.1 myc hisa

Transcriptional start site of human IRF-3 promoter was set as +1. The truncated human IRF-3 promoter plasmids pGL3-982, pGL3-149, and pGL3-67 were constructed as before [9 (link)]. The software Matlnspector (http://www.genomatix.de//matinspector.html) were used to search for IRF-3 promoter sequence for potential transcription factors target sites which are subject to acetylation. Deletion of pGL3-149 (pGL3-Del) was generated by PCR using the Site-Directed Mutagenesis Kit (Takara). Primer sequence for mutation were synthesized as 5’-GGCCCAGCGTAGAAAGGGCGGAACGCT-3’ (sense); 5’-ACCCGGCCCAGTGCGCAGGCGCG-3’ (anti-sense). All constructs were confirmed by sequencing without coding frame shifts. The plasmid pcDNA-p300 for overexpression experiment was a kind donate from Dr. Tony Kouzarides (The Gurdon Institute, UK) and the corresponding control plasmid pcNDA3.1/Myc-HisA was purchased from Invitrogen (Carlsbad, CA, USA).

As we described before, transcriptional start site (TSS) of human IRF-3 promoter was set as +1 and the IRF-3 genomic DNA fragment −982 to +18, −149 to +18, −67 to +18 was inserted into the pGL3-Basic vector (Promega), respectively named as pGL3-982, pGL3-149, and pGL3-67^{15 (link)}. The software TFSEARCH ver.1.3 (http://www.cbrc.jp/research/db/TFSEARCH.html) and Matlnspector (http://www.genomatix.de//matinspector.html) were used to search of consensus potential transcription factors target sites in IRF-3 promoter sequence. The GATA-1 binding site deletion promoter (pGL3-Del) was created by PCR from the cloned pGL3-149 plasmid, using site-directed mutagenesis kit (Takara). The forward primer was 5′-GGCCCAGCGTAGAAAGGGCGGAACGCT-3′; the reward primer was 5′-ACCCGGCCCAGTGCGCAGGCGCG-3′. For over-expression experiments, the plasmid pcDNA-GATA-1 was a kind gift from Dr. Feng Li (Li et al., 2015) and the corresponding control plasmid pcNDA3.1/Myc-HisA was purchased from Invitrogen (Carlsbad, CA, USA). The double-stranded siRNAs against human GATA-1 and control siRNA were synthesized and high-performance purified (GenePharma). The siRNA sequences used are the following (sense): 5′-AGUUGAGGCAGGGUAGAGCtt-3′ for GATA-1 siRNA; 5′-UUCUCCGAACGUGUCACGUtt-3′ for control siRNA.