Fig. 3. DCLK1 regulates NF-κB activity, independent of MAP7D1 and DCX.

A RAW264.7 cells were transfected to express NFκB-RE-EGFP reporter. Cells were then treated with 10 μM D-IN for 30 min before exposure to 0.5 μg/mL LPS for 2 hours. NF-κB reporter activity was measured by flow cytometry. B NF-κB reporter RAW264.7 cells were transfected with DCLK1 siRNA or control siNC. Following exposure to 0.5 μg/mL LPS for 2 hours, NFκB activity was measured. C NF-κB reporter RAW264.7 cells were transfected with DCLK1 cDNA plasmid or empty plasmid and exposed to 0.5 μg/mL LPS for 2 hours to measure NF-κB activity. D RAW264.7 cells were pretreated with 10 μM D-IN for 30 minutes or transfected with DCLK1 siRNA. Cells were then exposed to 0.5 μg/mL LPS for 2 hours. Proteins levels of IκBα and p65 were measured. Nuclear fractions were also probed for p65 protein. GAPDH was used as loading control for total cell lysates or cytosolic fractions. Lamin B was used as loading control for nuclear fractions. E RAW264.7 cells were treated as indicated in D. Cells were then fixed and stained for p65 subunit (green). DAPI (blue) was used to counterstain. F RAW264.7 cells transfected with MAP7D1 siRNA were exposed to 0.5 μg/mL LPS for 2 h. Proteins were probed for levels of IκB-α and p65 in cytosol and p65 in nuclear fractions. GAPDH and Lamin B were used as loading control respectively. G, H mRNA levels of Tnfa and Il6 in RAW264.7 cells transfected with MAP7D1 siRNA prior to LPS challenge [n = 3; Mean ± SEM; ns = not significant; ***p < 0.001]. I RAW264.7 cells transfected with DCX siRNA were exposed to 0.5 μg/mL LPS for 2 h. Proteins were probed for levels of IκB-α and p65 in cytosol and p65 in nuclear fractions. GAPDH and Lamin B were used as loading control respectively. J, K mRNA levels of Tnfa and Il6 in RAW264.7 cells transfected with DCX siRNA prior to LPS challenge [n = 3; Mean ± SEM; ns = not significant; ***p < 0.001].