Fig. 2

25KbPEI increases IFN-γ production by activating ERK/mTOR signaling pathways. A Immunoblot analysis conducted to detect phosphorylation of ERK and mTOR signaling pathway molecules at the indicated times after 25KbPEI treatment of NK-92MI cells (left). The bands were quantified using Image Lab, and the ratio of p-ERK/total ERK or p-mTOR/total mTOR is presented (right). Statistical analysis was conducted using two-way ANOVA with Sidak’s multiple comparisons test (vs. 0 h). B, C NK-92MI cells were pretreated for 1 h with U0126 (20 μM) or rapamycin (50 nM) and then incubated with 25KbPEI for 12 h. IFN-γ levels in the cytosol were measured by flow cytometry. D, E NK-92MI cells were pretreated with U0126 (20 μM) or rapamycin (50 nM) for 1 h, followed by 25KbPEI for 3 h. Then, p-mTOR or p-ERK levels in NK-92MI cells were measured by flow cytometry. Statistical analysis was conducted using one-way ANOVA with Tukey’s multiple comparisons test. F Schematic diagram showing the role of ERK and mTOR signaling in production of IFN-γ by 25KbPEI-treated NK-92MI cells. All experiments were conducted at least three times. *P < 0.05; **P < 0.01; ***P < 0.001, and ****P < 0.0001