American Society of Hematology

Figure 5

$Figure 5. Wogonoside promotes translocation of PLSCR1 into the nucleus and facilitates its binding to the IP3R1 promoter. (A) The cytoplasmic/membrane and nuclear fractions of the cells were analyzed by western blotting for the PLSCR1 protein, with β-actin and lamin B as cytoplasmic and nuclear loading controls, respectively. (B) U937 and HL-60 cells were incubated with wogonoside (150 μM) for 48 hours. Cells were then incubated with primary anti-PLSCR1 antibody (1:50) at 37°C for 1 hour and then at 4°C overnight, followed by incubation with FITC-labeled secondary goat anti-rabbit antibody (1:100) for 1 hour at 37°C. The coverslips were washed and counterstained with DAPI working solution (100 μg/mL) for 20 minutes. PLSCR1 antigen (green fluorescence) and cell nuclei stained with DAPI (blue fluorescence) were detected by confocal microscopy (FV1000; Olympus, Tokyo, Japan) with FV10-ASW2.1 acquisition software (Olympus) at room temperature. (Original magnification ×1000; immersion objective ×100 with immersion oil type F). Images are representative of 3 independent experiments. (C) EMSA assay to detect PLSCR1 binding to its consensus site in the IP3R1 promoter is shown. Cells were incubated with wogonoside (150 μM) for 48 hours, and DNA binding was determined in nuclear extracts using EMSA. To determine the composition of the DNA-binding complex, the anti-PLSCR1 antibody was used for supershift experiments. These figures are representative of 3 separate experiments. (D) The effect of wogonoside on IP3R1 expression was analyzed by western blotting.$

Wogonoside promotes translocation of PLSCR1 into the nucleus and facilitates its binding to the IP3R1 promoter. (A) The cytoplasmic/membrane and nuclear fractions of the cells were analyzed by western blotting for the PLSCR1 protein, with β-actin and lamin B as cytoplasmic and nuclear loading controls, respectively. (B) U937 and HL-60 cells were incubated with wogonoside (150 μM) for 48 hours. Cells were then incubated with primary anti-PLSCR1 antibody (1:50) at 37°C for 1 hour and then at 4°C overnight, followed by incubation with FITC-labeled secondary goat anti-rabbit antibody (1:100) for 1 hour at 37°C. The coverslips were washed and counterstained with DAPI working solution (100 μg/mL) for 20 minutes. PLSCR1 antigen (green fluorescence) and cell nuclei stained with DAPI (blue fluorescence) were detected by confocal microscopy (FV1000; Olympus, Tokyo, Japan) with FV10-ASW2.1 acquisition software (Olympus) at room temperature. (Original magnification ×1000; immersion objective ×100 with immersion oil type F). Images are representative of 3 independent experiments. (C) EMSA assay to detect PLSCR1 binding to its consensus site in the IP3R1 promoter is shown. Cells were incubated with wogonoside (150 μM) for 48 hours, and DNA binding was determined in nuclear extracts using EMSA. To determine the composition of the DNA-binding complex, the anti-PLSCR1 antibody was used for supershift experiments. These figures are representative of 3 separate experiments. (D) The effect of wogonoside on IP3R1 expression was analyzed by western blotting.

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