Figure 2.

Stat3 and NF-κB (p65) nuclear interaction is enhanced in irradiated human glioma xenograft cell lines. (A) The cytoplasmic and nuclear extracts from IR-treated (0 to 8 Gy) 4910 and 5310 cells were subjected to WB. Blots were reprobed with antibodies against GAPDH and HDAC-1 to monitor equal loading of cytoplasmic and nuclear fractions, respectively. (B) The control and IR-treated cells were immunostained to check the expression and sub-cellular distribution of p65 (red) and Stat3 (green). DAPI was used for nuclear counterstaining and representative pictures from three individual experiments were presented. (C) Co-IP experiments were carried out by precipitating the NEs (500 μg) with non-specific IgG and p-Stat3 antibodies using μMACS^™ protein G microbeads and MACS separation columns following the manufacturer’s instructions. WB was carried out using immunoprecipitates. Blots were stripped and reprobed with antibody used for IP to confirm the antibody specificity. Input control samples (50 μg of pre-immunoprecipitated samples) were subjected to IB and input blots were also probed with HDAC-1 to monitor equal loading. (D) IP was performed using p-p65 antibody and immunopecipitates were subjected WB as described above. (E) IP experiments were carried out with ace-p65 antibody. Representative blots from three independent experiments were presented.