Fig. 6.

OT-mediated expression and clustering of NF-κB/CREB1/CBP complex promotes Cx43 expression. A, Mouse ESC were treated with OT(10⁻⁷ m) for different time periods (0–120 min). Cell lysates were immunoblotted with anti-CREB1, anti-CBP, anti-β-actin, or antilamin A/C antibody. Each example shown is representative of five independent experiments. The lower part (A) depicting the bars denotes the mean ± se of five independent experiments for each condition determined from densitometry relative to β-actin. *, P < 0.05 vs. control. B, Immunofluorescence confocal microscopy in combination with in situ PLA, which detects protein-protein complexes, was used to explore interactions between phospho-NF-κB, CREB1, and CBP. Each detected complex is represented by a green dot. Nuclei were counterstained by PI (red). Scale bars, 20 μm (magnification, ×400). C, Mouse ESC were pretreated in the absence and presence of SN 50 (500 ng/ml) or bay 11–7082 (10⁻⁵ m) for 30 min before 90 min OT treatment. Cell lysates were analyzed by Western blotting with antibodies that recognize phospho-NF-κB, CREB1, and CBP. Immunoprecipitation of phospho-NF-κB was analyzed by Western blotting with antibodies that recognize CREB1 and CBP. Each of the examples is representative of five independent experiments. The right part (A) depicting the bars denotes the mean ± se of five independent experiments for each condition determined from densitometry relative to β-actin. *, P < 0.05 vs. control. D, Mouse ESC were transfected for 24 h with either CREB1-specific siRNA (100 nmol/liter) or nontargeting control siRNA (100 nmol/liter) using Hyperfectamine before 1 h OT treatment. CREB1 expression was analyzed by Western blot. Each of the examples shown is representative of four independent experiments. E, Mouse ESC were transfected for 24 h with either CBP-specific siRNA (100 nmol/liter) or nontargeting control siRNA (100 nmol/liter) using Hyperfectamine before 1 h OT treatment. CBP expression was analyzed by Western blot. Each of the examples shown is representative of four independent experiments. F, Mouse ESC were transfected for 24 h with either CREB1-specific siRNA, CBP-specific siRNA, or nontargeting control siRNA before OT (10⁻⁷ m) treatment for 3 h, after which Cx43 protein expression levels were detected. Each example shown is representative of four experiments. The lower part (D–F) depicting the bars denotes the mean ± se of four independent experiments for each condition determined from densitometry relative to β-actin. *, P < 0.05 vs. control; **, P < 0.05 vs. OT alone. Con, Control; ntRNA, nontargeting RNA; ROD, Relative Optical Density.