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. 2017 Nov 23;9(12):355. doi: 10.3390/v9120355

Figure 2.

Figure 2

The DLG1-Tax co-localization pattern does not arise from an overabundance of PDZ domains inside the cell. (A) Schematic representation of DLG1 and PAR3 proteins, showing that both proteins bear three PDZ domains. SH3, interaction domain Src homology 3; GK, homologous to the yeast guanylate kinase enzyme; CR1, oligomerization domain; aPKCBD, atypical protein kinase C binding domain; (B) Subcellular distribution of the PAR3 PDZ-containing protein and Tax in epithelial cells. A plasmid encoding seyfp2 tagged PAR3 or mTurq2 tagged Tax was transfected in HEK293 cells and its expression was analyzed by confocal microscopy (red); (C) Simultaneous overexpression of Tax and PAR3 proteins in epithelial cells. The plasmids encoding seyfp2-PAR3 (red) and mTurq2-Tax proteins (green) were co-transfected in HEK293 cells and co-localization between both proteins was tested. In (B,C), the white arrows indicate the expression of the over-expressed proteins as described in the text. All scale bars are 5 µm; (D) Influence of Tax expression over ectopic DLG1 and PAR3 subcellular distribution. HEK293 cells transfected with pseyfp2-Tax or with the control empty vector pseyfp2-N1 together with the pmTurq2-DLG1 or pseyfp2-PAR3 expressing plasmids were harvested, and detergent soluble and insoluble fractions were separated by centrifugation. The mTurq2-DLG1, seyfp2-PAR3, and seyfp2-Tax expression levels in each fraction were assessed by Western blot using anti-green fluorescent protein (anti-gfp) antibody; (E) Influence of Tax expression over endogenous DLG1 and PAR3 subcellular distribution. HEK293 cells transfected with pseyfp2-Tax or with the control empty vector pseyfp2-N1 were harvested, and detergent soluble and insoluble fractions were separated by centrifugation. Endogenous DLG1 and PAR3 expression levels in each fraction were assessed by Western blot using anti-DLG1 and anti-PAR3 antibodies. The seyfp2-Tax expression was ascertained using an anti-gfp antibody. For (C,D), the γ-tubulin and Transferrin receptor (TR) expression, which were detected with anti-γ-tubulin and anti-TR antibodies, were used as loading control; (F) Influence of Tax expression over DLG1 total levels. HEK293 cells transfected with pseyfp2-Tax or with the control empty vector pseyfp2-N1 were harvested, and total protein extracts were separated by SDS-PAGE. The endogenous DLG1 expression level was assessed by Western blot using anti-DLG1, and seyfp2-Tax expression was ascertained using an anti-gfp antibody.