MEC1 cells were taken from the culture, centrifuged and washed with PBS two times, and then resuspended in 100 µl of PBS. The cell suspension was mixed with 10 µl of 2 µg/µl plasmid and electroporated using the Neon transfection system (Invitrogen), using the pulse setting 1,200/20/2 (pulse voltage/pulse width/number of pulses). After electroporation, the cells were cultivated overnight in RPMI supplemented with 20% FBS. For the visualization of endocytic machinery, we used plasmid GFP-rab11 WT (Addgene, cat. no. 12674) (Choudhury et al., 2002 (link)).
Gfp rab11 wt
Manufactured by Addgene
GFP-rab11 WT is a fluorescently-labeled protein construct that consists of the GFP (green fluorescent protein) tag fused to the wild-type Rab11 protein. Rab11 is a small GTPase that regulates the recycling of proteins and membranes within the cell. The GFP tag allows for the visualization and tracking of Rab11 localization and dynamics within live cells.
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Lab products found in correlation
2 protocols using gfp rab11 wt
1
Transfection of MEC1 Cells with GFP-Rab11
2
Plasmid Construction and Characterization
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Backbone of the plasmid pcDNA4-TO-RNF43-2xHA-2xFLAG (kindly gifted by Bon-Kyoung Koo together with pcDNA4-TO-RNF43Mut1-2xHA-2xFLAG (Koo et al., 2012) (link) Other plasmids used were described previously and included: myc-Vangl1, GFP-Vangl2, GFP-Vangl2ΔN, GFP-Vangl2ΔC, GFP-Vangl2ΔNΔC (Belotti et al., 2012) (link), pEGFP-C1-Rab5a (Chen et al., 2009) (link), GFP-rab11 WT (Addgene #12674), His-Ubiquitin (Tauriello et al., 2010) (link), pcDNA3-Flag-mDvl1 (Tauriello et al., 2010) (link), pCMV5-3xFlag Dvl2 (Addgene #24802), pCDNA3.1-Flag-hDvl3 (Angers et al., 2006) (link) Finally, PCR products were sequenced using the Illumina platform and compared with the reference sequence (Malcikova et al., 2015) (link). Sequencing results are presented in the Supplementary Table 1.
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