HA-tagged GFP (pcDNA3.1-3XHA-GFP plasmid; Brady et al., 2011 (link)) and FLAG (DYKDDDDK)-tagged hRhoD ORF clone (pcDNA3.1+C-(K)-DYK, ABIN4924331, Genomics-Online.com) overexpression constructs were transfected into sgp53(2) E1A;HrasG12V;H11Cas9 MEFs using Lipofectamine 2000 (Invitrogen), as described above. After 24 h, cells were harvested, fixed, permeabilized, and stained with Alexa Fluor 647 Phalloidin (A22287; Thermo Fisher Scientific, 1:1,000) to mark F-actin, mouse anti-FLAG (clone M2 F1804; Sigma-Aldrich, 1:50), mouse anti-HA (12CA5; Sigma-Aldrich, 1:50), fluorescein goat anti-rabbit IgG (FI-1000; Invitrogen, 1:250), and fluorescein anti-mouse IgG (FI-2000; Invitrogen, 1:250). Cells were visualized using a Leica DMi8 inverted microscope with a 63×/1.4 oil-immersion objective. Z-stack images were captured and processed using a Leica DFC9000 GT digital camera, LASX acquisition software, and 3D-deconvolution and maximum-projection algorithms.
Protocol detail
Fluorescent Protein Overexpression in Engineered MEFs
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Lipofectamine 2000
Alexa Fluor 647 Phalloidin (A22287;
mouse anti-FLAG (clone M2 F1804;
mouse anti-HA (12CA5;
DMi8 inverted microscope
Alexa fluor 647
Anti igg
Camera
Cells
Clone
F actin
Fluorescein
Goat
Immersion
Lipofectamine 2000
Microscope
Mouse
Phalloidin
Plasmid
Rabbit
Corresponding Organization :
Other organizations : Stanford University, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, University of Rochester Medical Center, Howard Hughes Medical Institute, The University of Texas Southwestern Medical Center, Children's Medical Center
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Variable analysis
independent variables
- Overexpression of HA-tagged GFP (pcDNA3.1-3XHA-GFP plasmid)
- Overexpression of FLAG (DYKDDDDK)-tagged hRhoD ORF clone (pcDNA3.1+C-(K)-DYK, ABIN4924331)
- Localization/distribution of HA-tagged GFP
- Localization/distribution of FLAG-tagged hRhoD
- Cell line: sgp53(2) E1A;Hras^G12V;H11^Cas9 MEFs
- Transfection method: Lipofectamine 2000
- Fixation and permeabilization of cells
- Staining with Alexa Fluor 647 Phalloidin, anti-FLAG antibody, and anti-HA antibody
- Visualization using Leica DMi8 inverted microscope and image processing techniques
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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