PC12-GR5 cells were maintained at 37°C and 5% CO2 in T75 flasks (Sarsdtedt 83-1813-002) in dye-free DMEM (Life Technologies 21063-029) supplemented with 5% fetal bovine serum and 5% horse serum. Transfections were performed with Lipofectamine 2000 (Invitrogen) and 2 μg of plasmid after one day of growth on coverslips. The myristoylated psCFP2 expression constructed was made by PCR amplification of the psCFP2 gene (Evrogen) including the mammalian codon optimized myristoylation signal peptide from the v-src gene: MGSSKSKPKDPSQRRNNN. The amplicon was subsequently cloned into the empty N1 mammalian expression vector (Clontech) using the BglII (5′) and NotI (3′) restriction sites. pEGFPC1-Epsin1 was obtained from Addgene (#22228). Epsin1-mcherryC1 and Epsin1-mEos3 were both made by replacing EGFP from pEGFPC1-Epsin1 with mCherry (Clontech #632524) or mEos319 (link) using Age1 and BsrG1. For live imaging, cells were transfected with 1 μg each of pEGFP-LCa (clathrin light chain a)20 (link) and pmCherryC1-Epsin1.
Empty n1 mammalian expression vector
Manufactured by Takara Bio
The Empty N1 mammalian expression vector is a plasmid designed for the expression of recombinant proteins in mammalian cell lines. It provides a backbone for cloning and expressing genes of interest in a eukaryotic system.
Automatically generated - may contain errors
2 protocols using empty n1 mammalian expression vector
1
Visualization of Epsin1 and Clathrin Dynamics
2
Visualization of Epsin1 and Clathrin Dynamics
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PC12-GR5 cells were maintained at 37°C and 5% CO2 in T75 flasks (Sarsdtedt 83-1813-002) in dye-free DMEM (Life Technologies 21063-029) supplemented with 5% fetal bovine serum and 5% horse serum. Transfections were performed with Lipofectamine 2000 (Invitrogen) and 2 μg of plasmid after one day of growth on coverslips. The myristoylated psCFP2 expression constructed was made by PCR amplification of the psCFP2 gene (Evrogen) including the mammalian codon optimized myristoylation signal peptide from the v-src gene: MGSSKSKPKDPSQRRNNN. The amplicon was subsequently cloned into the empty N1 mammalian expression vector (Clontech) using the BglII (5′) and NotI (3′) restriction sites. pEGFPC1-Epsin1 was obtained from Addgene (#22228). Epsin1-mcherryC1 and Epsin1-mEos3 were both made by replacing EGFP from pEGFPC1-Epsin1 with mCherry (Clontech #632524) or mEos319 (link) using Age1 and BsrG1. For live imaging, cells were transfected with 1 μg each of pEGFP-LCa (clathrin light chain a)20 (link) and pmCherryC1-Epsin1.
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