Cells were transfected with small interfering RNA (siRNA) or plasmids three days before the experiments using a Nucleofector ™ System (Amaxa Biosystems) set to the U25 program. To downregulate Nox4 or SSH1L phosphatase, 3 μg custom designed siRNA against each human sequence (5′-GCAUCUGUUCUUAACCUCA-3′ for Nox4 and 5′-ACUGAGGUACAGCUGGAUGUU-3′ for SH1L) was used to electroporate one million cells. All Stars negative control siRNA (Qiagen 1027281) was used as a control siRNA. To increase cofilin activity, cells were co-transfected with siRNA (negative control or against Nox4) and either 1.1 μg of a plasmid containing a constitutively active form of cofilin (GFP_S3A_cofilin, kind gift of Dr. Condeelis, Albert Einstein College of Medicine, NY) or 1.0 μg of GFP-empty vector as a control. Similarly, in order to intensify LIM kinase activity, cells were transfected with 2 μg of an EGFP-plasmid encoding a constitutively active LIM kinase mutant [6 (link)] or EGFP empty vector (Clontech) as a control.
Egfp empty vector
Manufactured by Takara Bio
The EGFP empty vector is a plasmid that contains the enhanced green fluorescent protein (EGFP) gene without any additional DNA sequences. It serves as a control vector for experiments involving EGFP expression and fluorescence detection.
Automatically generated - may contain errors
2 protocols using egfp empty vector
1
Modulating Actin Dynamics via siRNA and Plasmids
2
Quantifying Exonuclease Activity via qPCR
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To examine the enzyme activity of ExoIII or T7, we utilized real-time quantitative PCR using a fluorescent reporter, SYBR Green I (Thermo Fisher Scientific, MA, USA). SYBR Green I has been used in a variety of quantitative dsDNA detection and analysis techniques. Briefly, the eGFP empty vector (Clontech) was linearized with EcoRI (leaving a 5′ protruding end), SmaI (leaving a blunt end) or KpnI (leaving a 3′ protruding end). The linearized eGFP empty vectors were then mixed with SYBR Green I, followed by treatment with ExoIII or T7. Each well contained 10 μg eGFP empty vectors and 20 units enzyme/well in a 20 μl reaction mixture. The vectors were treated with ExoIII at 37 °C or T7 at 30 °C for 50–60 min. The digestion activities of ExoIII and T7 were estimated by the reduction in fluorescence intensity, which was measured every minute with an Applied Biosystems® 7500 real-time PCR system (Thermo Fisher Scientific, MA, USA). We performed five independent experiments and averaged the resulting data. To estimate the overhang lengths of the targeting vectors that were transfected into ES cells or fertilized mouse eggs, we repeated the same experiments with those vectors.
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