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. 2018 Dec 21;20(1):19. doi: 10.3390/ijms20010019

Figure 2.

Figure 2

Figure 2

In vitro testing of conditional expression of human Siglec-8 in transfected HEK 293T cells. (A) ROSA26-CMV-Siglec-8 and ROSA26-CAG-Siglec-8 conditional knock-in targeting constructs were transfected with or without the Cre:GFP plasmid into confluent HEK 293T cells. Twenty-four hours after transfection, cells were examined and photographed under fluorescent microscope. The presence of GFP indicated successful transfection and expression of the Cre:GFP plasmid; (B) total cellular proteins from each sample were harvested and subjected to 4–12% gradient SDS-PAGE and analyzed by western blot using anti-Siglec-8 antibody (Sheep, primary). Anti-sheep IgG conjugated with AP (secondary) was used to visualize the positive bands; (C) transfected HEK 293T cells were analyzed by flow cytometry using anti-Siglec-8 antibody. Only cells transfected with ROSA26-CAG-Siglec-8 or ROSA26-CMV-Siglec-8 construct plus the Cre:GFP plasmid showed Siglec-8 expression.