The ORF of Flot2 was cloned into a BamHI- and NotI-digested pEF1/myc-His B vector (Invitrogen), which is a 6.2-kb expression vector used to overexpress recombinant protein. pFlag-CMV-3 (Sigma, Rockford, IL, USA) is a 6.2-kb expression vector transiently or stably expressed in mammals, in which the N-terminal Flag can form fusion protein with a correctly inserted ORF. The ORF of PLCD3 was amplified from 5–8F cDNA by PCR using the sense primer (5′-CAAGCTTATGCTGTGCGGCCGCTGGA-3′) and the antisense primer (5′-CGGATCCTCAGGAGCGCTGGATGCGGAT-3′), and then subcloned into pFlag-CMV-3. Then, pEF1/myc-His-Flot2 and pFlag-CMV-PLCD3 were transfected into 293T cells with Lipofectamine 2000™ (Invitrogen). All experiments were performed according to a previously published protocol (38 (link)). Anti-Flag (Sigma) was used for immunoprecipitation. Anti-His (ProteinTech, Wuhan, China) was used for immunoblotting.
Pflag cmv
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PFlag-CMV is a plasmid vector designed for recombinant protein expression in mammalian cell lines. It contains a CMV promoter and a FLAG epitope tag for detection and purification purposes. The core function of this product is to enable the expression and detection of target proteins in a mammalian cell system.
Automatically generated - may contain errors
2 protocols using pflag cmv
1
Overexpression of Flot2 and PLCD3
2
Immunoprecipitation of Flotillin-1 and Flotillin-2
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pEF1/myc-His-Flot-1 and pFLAG-CMV-Flot-2 expression vectors were constructed by cloning the Flot-1 ORF and Flot-2 ORF into pEF1/myc-His vector (Invitrogen, USA) and pFLAG-CMV vector (Sigma, USA), respectively. The pEF1/myc-His-Flot-1 and pFLAG-CMV-Flot-2 vectors were transfected into 293T cells in different combinations. Forty-eight hours later, cells lysates were prepared and pre-incubated with agarose IgA/IgG beads for 2 h at 4 °C. Then, beads were removed and fresh agarose IgA/IgG beads with anti-His or anti-Flag were incubated with the lysates overnight at 4 °C. After washing, denaturation, and SDS-PAGE, the proteins were visualized by immunoblotting. The endogenous interactions in 5-8F cells were analyzed in a similar way with anti-Flot-1. The experiment was repeated for three independent times.
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