Figure 4 | Scientific Reports

Figure 4

From: IRGM1 enhances B16 melanoma cell metastasis through PI3K-Rac1 mediated epithelial mesenchymal transition

Figure 4

IRGM1 activates PI3K-Rac1 signaling pathway.

B16 cells were transfected with either control or IRGM1siRNA (a) or transduced with either GFP or IRGM1-GFP lentiviral vectors (b–h). (a,b) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. (c) Co-localization of PIK3CA with IRGM1 was determined by immunofluorescence staining in IRGM1 overexpressing B16 cells (Upper panel: GFP; lower panel: IRGM1-GFP; green: IRGM1-GFP/GFP; red: PI3KCA; blue: nuclei). (d) Co-IP study was performed to further confirm the co-localization of IRGM1 and PIK3CA. Anti-IRGM1 polyclonal antibody or non-immune IgG (control) were used to pull down IRGM1 from total cell lysates. Anti-PIK3CA monoclonal antibody was used to detect participated PIK3CA. GFP and IRGM1 overexpressing B16 cells were transfected with either control or PIK3CA siRNA (e–h). (e) PIK3CA levels were measured by western blot. (f) Rac1 activity was measured in total lysates by GST-PAK-PBD pull down and Rac1 levels were measured by western blot. The migration (g) and invasion (h) ability of GFP and IRGM1 overexpressing B16 cells were evaluated by using boyden chamber. Data represent three independent experiments. (Unpaired t-test; **p < 0.01, ***p < 0.001)

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