Pib v5 his vector

The pIB-AcerOr1/pIB-AcerOr2 plasmid constructs containing intact open reading frames, which were amplified with specific primers containing BamHI and EcoRI (NEB, Beverly, MA, USA) sites for the A. cerana cerana ORs AcerOr1 and AcerOr2 and cloned in the multiple cloning site of the pIB/V5-His vector (Invitrogen, Carlsbad, CA, USA), were used to generate the final transformation plasmids by restriction digestion with BamHI and EcoRI (NEB, Beverly, MA, USA). The specific primer sequences were as follows: AcerOr1 F: 5′-CGCGGATCCATGGAAAATACCACGAATTATCGTA-3′, AcerOr1 R: 5′-CCGGAATTCTACCGTCATTGCACGCAGAA-3′, AcerOr2 F: 5′-CGCGGATCCATGATGAAGTTCAAGCAACAGGG-3′, AcerOr2 R: 5′-CCGGAATTCCTTCAGTTGCACCAACACCA-3′.

The coding regions of human wild-type K-Ras and H3 were amplified by PCR and were subcloned into pEGFP-N1 plasmid (Clontech Inc., Palo Alto, CA). The amplified PCAF and HDAC2 were subcloned into pcDNA6.0/HA-tag vector (Invitrogen, Carlsbad, CA, USA). The pEGFP-K-Ras^G12V/T35S construct was mutated using site-directed mutagenesis. The pEGFP-H3K9Q construct was constructed using the TaKaRa MutanBEST Kit (#D401, TaKaRa, Dalian, China). Two different sequences of siRNAs specific for HDAC2 (si-HDAC2–1 and si-HDAC2–2) were purchased from GenePharma (Shanghai, China). A non-targeting sequence was used as a negative control (si-con). MDM2-MU for expression of MDM2^C464A was constructed and recombination into pIB/V5-His Vector (Invitrogen).
SW48 cells were seeded in 6-well plates with a density of 1 × 10⁵ cells/well. When 50% confluence was researched, the cells were transfected with plasmids or siRNAs by using lipofectamine 3000 (Invitrogen). At 48 h of transfection, the culture medium was replaced by the complete medium to stop transfection. Transfection efficiency was confirmed by using Western blot and/or RT-qPCR.

Sf9 cells were cultured in an incubator (Thermo scientific, OH, USA) at a constant temperature of 24 to 25 °C. SF900-II media (Invitrogen, CA, USA) was used to culture cells. Sf9 cells were transfected with full coding sequences of cDNA of odorant receptors (AaOrco, AaOr8, and AaOr49) of Ae. aegypti, which were inserted into pIB/V5-His vector using Cellfectin® II Reagent (Invitrogen, Grand Island, NY, USA). Transfected cell lines were cultured in 10 μg/ml blastcidine SF900-II media (Invitrogen, Grand Island, NY, USA). Full length cDNAs were synthesized from mRNA extracted from the stylet with Superscript III (Invitrogen, CA, USA) using gene specific primers (Table S1). The expression vector plasmids were synthesized by inserting cDNAs of AaOrco, AaOr8, and AaOr49 into multiple cloning sites of a pIB/V5-His vector (Invitrogen, CA, USA) using the restriction enzymes NotI and XbaI (Koscamco, Anyang, Korea). Template pDNA for pIB-Or8 and Or49 and primer were then mixed with kit solutions and the total PCR reaction volume and conditions were followed as described earlier34 (link).