Figure 6. Effect of knockdown IκBα on the expression and cellular localization of NF-κBp65 in HCM cells 24 h, 48 h, and 72 h after IκBα siRNA transfection. A: NF-κBp65 mRNA expression in HCM cells of IκBα siRNA transfected, nonsense control siRNA (NC) transfected, and control was quantified by real-time RT-PCR. Expression levels were normalized with GAPDH. B: Nuclear proteins extracted from IκBα siRNA, nonsense control siRNA (NC), as well as control cells, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrow indicates the NF-κBp65 (75 kDa) band and nucleus internal control, Lamin B (67 kDa). The bands were analyzed densitometrically, and the values were normalized with Lamin B, represented in bar graph. C: Total cell lysates from HCM cells transfected with IκBα siRNA, and nonsense control siRNA (NC) as well as from the control, respectively, were analyzed by western blot with NF-κBp65 antibody. The arrows show the NF-κBp65 (75 kDa) band and the internal control, GAPDH (36 kDa). The mRNA and protein values compared to the control and nonsense control siRNA (NC) were determined by one-way ANOVA. The double asterisk denotes p<0.01. D: The HCM cells were immunostained with NF-κBp65 antibody and analyzed by fluorescence microscopy. A weak nuclear signal of NF-κBp65 (green) was observed in control cells at 24 h, 48 h, and 72 h. After IκBα siRNA transfection, NF-κBp65 translocated from the cytoplasm into the nucleus, and a strong signal of NF-κBp65 was detected in the nucleus at 24 h, 48 h, and 72 h. Cy3 labled IκBα siRNA (red) was observed in the cytoplasm. Cell nuclei were counterstained with DAPI (blue).