Role of PKD in TNF-α-induced NF-κB activation.
A, Caco2 cells were serum-starved for 3 h, treated with 10 μg/ml CID755673 or 50 μm CID2011756 in combination with 10 μm MG-132 for 30 min, and subsequently stimulated with 20 ng/ml TNF-α for the indicated times. IκB phosphorylation was measured as described in the legend to Fig. 3B. The averages of the intensities of the immunoreactive signals of phospho-IκB at 90 min of three independent experiments are expressed as the mean ± S.D. (error bars) in arbitrary units (AU) with p values (right). B, Caco2 cells were treated as described in A except that MG-132 treatment was omitted. Nuclear and cytoplasmic localization of p65 was measured as described in the Fig. 3A legend. TBP and α-tubulin were used as markers for the nuclear and the cytoplasmic fractions, respectively. The averages of the intensities of the immunoreactive signals of p65 in the nuclear fractions at 90 min of three independent experiments are expressed as the mean ± S.D. in arbitrary units with p values (right). C, Caco2 cells transfected with the indicated siRNAs were cultured for 38–42 h, serum-starved for 3 h, and stimulated with 20 ng/ml TNF-α for the indicated times. PKD phosphorylated at Ser-916, total PKD, PLCϵ, and actin in the cell lysates were detected by immunoblotting with the anti-phospho-PKD, anti-PKD, anti-PLCϵ, and anti-actin Abs, respectively. The numbers below the immunoblots indicate -fold changes of the phospho-PKD signals divided by the total PKD signals over that at 0 min after TNF-α stimulation of the control cells. The averages of the intensities of the immunoreactive signals of phospho-IκB at 90 min of three independent experiments are expressed as the mean ± S.D. in arbitrary units with p values (right). Three experiments performed independently yielded equivalent results.