Generation of stable CAV1 mutant cell lines

The plasmids pLacIOP (referred to as mock) and pLacIOP-caveolin-1 (referred to as CAV1, containing the full-length dog caveolin-1 sequence, NCBI Reference Sequence: NP_001003296.1) were previously described [42 (link), 90 (link)]. B16F10 cells were grown to 50-60% confluence in 6 multi-well plates and then transfected with 4 μg of pLacIOP-CAV1(Y14F) (referred as CAV1/Y14F) and pLacIOP-CAV1(Y14E) (referred as CAV1/Y14E), using the FuGene HD reagent, according to the manufacturer's indications. After transfection, cells were plated in complete RPMI medium containing hygromycin (750 μg/mL) for 2 to 3 weeks, to yield stably transfected B16F10(CAV1/Y14F) and B16F10(CAV1/Y14E) cells, respectively.

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Ortiz R., Díaz J., Díaz N., Lobos-Gonzalez L., Cárdenas A., Contreras P., Díaz M.I., Otte E., Cooper-White J., Torres V., Leyton L, & Quest A.F. (2016). Extracellular matrix-specific Caveolin-1 phosphorylation on tyrosine 14 is linked to augmented melanoma metastasis but not tumorigenesis. Oncotarget, 7(26), 40571-40593.

Publication 2016

HD reagent

Cav1 Cells Fugene Hygromycin Plasmids Transfection

Corresponding Organization :

Other organizations : Advanced Center for Chronic Diseases, University of Chile, Universidad Bernardo O'Higgins, Instituto de Neurociencia Biomédica, Fundación Ciencia and Vida, University of Queensland

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Variable analysis

independent variables

Transfection of B16F10 cells with pLacIOP-CAV1(Y14F) (referred as CAV1/Y14F) and pLacIOP-CAV1(Y14E) (referred as CAV1/Y14E)

dependent variables

Stable transfection of B16F10 cells to yield B16F10(CAV1/Y14F) and B16F10(CAV1/Y14E) cells

control variables

B16F10 cells grown to 50-60% confluence in 6 multi-well plates
Transfection performed using FuGene HD reagent according to manufacturer's instructions
Cells plated in complete RPMI medium containing hygromycin (750 μg/mL) for 2 to 3 weeks

controls

PLacIOP (referred to as mock) plasmid as a negative control

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