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. 2021 Dec 23;12:767366. doi: 10.3389/fimmu.2021.767366

Figure 1.

Figure 1

Syk was a prime activator of the NF-κB signaling pathway in macrophages. (A) RAW264.7 cells co-transfected with the NF-κB-Luciferase reporter gene plasmid and either empty (pcDNA), Syk, Src, or Akt plasmids were treated with either LPS (1 μg/mL), TNF-α (20 ng/mL), or PMA (100 nM) for 24 h. Luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. (B, C) Syk, p-Syk, p85, p-p85, IκBα, and p-IκBα in whole-cell lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analyses. (D) p65 and p50 in the nuclear lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (E) IκBα and p-IκBα in the whole-cell lysates of WT and Syk−/− RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by Western blot analysis. (F) p65 and p50 in the nuclear lysates of WT and Syk−/− RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time were detected by western blot analysis. (G) IκBα and p-IκBα in the whole-cell lysates of RAW264.7 cells and p65 and p50 in the nuclear lysates of RAW264.7 cells treated with LPS (1 μg/mL) for the indicated time in the absence or presence of Pic (50 μM) and Bay (10 μM) were determined by western blot analysis. (H) WT and Syk−/− RAW264.7 cells transfected with the NF-κB-luciferase reporter gene plasmids for 24 h were treated with LPS (1 μg/mL) for 24 h, after which the luciferase activity of these cells was measured by a luminometer and normalized to β-galactosidase activity. (I) HEK293 cells co-transfected with the NF-κB-Luciferase reporter gene plasmids and Myc-Syk plasmids for 24 h were treated with PMA (100 nM), Pic (0–50 μM), and Bay (0-10 μM), and the luciferase activity was measured by a luminometer and normalized to β-galactosidase activity. *P < 0.05, **P < 0.01 compared with controls.