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. 2015 Dec 1;6:1067. doi: 10.3389/fpls.2015.01067

FIGURE 1.

FIGURE 1

Subcellular targeting strategies tested for stabilize TG2 in leaves. (A) Schematic representation of the TG2 constructs used for Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves. Cyto-TG2 is a cytosolic form of TG2. Sec-TG2, ER-TG2, and Vac-TG2 are introduced in the secretory pathway with murine signal peptide (SP) from gamma 1 antibody chain; SEKDEL, ER retention SP; KISIA is a CT vacuolar targeting signal of the amaranth 11S globulin. Scheme is not drawn to scale. (B) Enzyme-linked Immunosorbent Assay (ELISA) of TG2 fused to the different sorting signals. Microwells were coated with the same amount of total leaves extract overnight at 4°C. After blocking, anti-TG2 mAb 2G3 was added, followed of incubation with a biotin-conjugated anti-mouse, later with HRP-conjugated streptavidin and developed with TMB peroxidase substrate. Three biological replicates (each replicate containing five leaf disks of the infiltrated tissue from a different plant) were used for ELISA. Error bars represent the standard error of the mean (SEM). ∗∗∗∗Denotes statistically significant difference by Tukey’s multiple comparisons test (P < 0.001). (C) Western blot of TG2 fused to the different sorting signals. Expression levels were measured by scanning densitometry of Western Blot developed with 2G3 mAb with a minimum of three independent experiments. The amount of total extract was adjusted using RLS stained with Coomassie Brilliant Blue R-250 as loading control.