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. 2018 Jun 8;2(3):032001. doi: 10.1063/1.5019770

FIG. 2.

FIG. 2.

Lentiviral expression system for constitutive expression of MscL G22S in MDA-MB-231 cells. (a) A single lentivirus vector system for bicistronic expression of cytosolic EGFP and MscL from a single promoter. EGFP and MscL genes are encoded with a P2A linker sequence in between. Protein translation results in an incomplete peptide bond of the P2A linker's final amino acid, resulting in the expression of separate EGFP and MscL proteins. (b) Western blot analysis of transduced whole cells with a negative control vector, no MscL EGFP-only, and experimental cells, EGFP-P2A-MscL G22S with the periplasmic FLAG-tag. GAPDH was used as a housekeeping protein. (c) Flow cytometry fluorescence analysis using anti-FLAG Alexa Fluor® 647 of methanol fixed and permeabilized cells (left) and PFA fixed cells for surface analysis (right). Negative controls were EGFP-P2A-MscL G22S cells with no anti-FLAG and no MscL EGFP-only cells with anti-FLAG, and experimental cells were EGFP-P2A-MscL G22S with anti-FLAG. (d) Immunostaining of FLAG for no MscL EGFP-only cells (top) and EGFP-P2A-MscL G22S with FLAG-tag (bottom) with methanol permeabilization and fixation (2 leftmost panels) and PFA fixed cells for surface analysis (2 rightmost panels). DAPI was used to label cell nuclei. Scale bar = 25 μm.