Figure 4. DRAIC knockdown increases NF-κB activity.

(A) LNCaP cells were transfected with two different concentrations of siRNA (25 and 50 nM) against DRAIC or negative control (NC), followed by 10 μM MG132 treatment for 4 hours. Cell lysates prepared using RIPA buffer and immunoblotted for phospho IκBα, total IκBα and α-Tubulin. (B, C) HeLa cells were stably transfected with EV (empty vector) or FL (expressing full length DRAIC) and RT-qPCR performed to measure DRAIC over-expression. Mean± s.d, n = 3, *P<0.05. (C) HeLa cells from (B) were treated with TNF-α followed by 10 μM MG132 for 4 hours and cell lysates immunoblotted for phospho IκBα, total IκBα and α-Tubulin. (D) LNCaP cells transfected with NC or siRNA against DRAIC. RT-qPCR of indicated NF-κB responsive genes, expressed after normalization to 18S RNA and then to level of expression in NC cells. Mean ± s.d, n = 3, *P<0.05. (E) ChIP-qPCR of p65 at sites known in the literature to bind NF-κB at promoters of NF-κB responsive genes. IgG ChIP is taken as 1. Mean± s.d, n = 3, *P<0.05. (F) Matrigel invasion assay performed in LNCaP cells treated with either siRNA NC or against DRAIC (si787). DRAIC depleted cells were treated with IKK inhibitor, Bay11–7082 (6 hr) or sip65 (48 hr). Scale bar 20 μm. (G) LNCaP cells transfected with siRNAs immunoblotted for p65 and Tubulin. (H) The invasive cells in (F) counted under microscope by taking 10 random fields. Results expressed as mean ± s.d, n = 3, *P<0.05. *P<0.05 relative to NC.