Nuclear translocation of STAT3 and NF-κB. (A-B) Immunofluorescent staining of (A) STAT3-P^S727 and (B) NF-κB in cells before and after cell culture for 1 or 2 weeks. Slides were costained with mouse (A) STAT3-P^S727 or (B) NF-κB antibody, showing the red color and a rabbit anti-HMGB1 antibody, showing green. Yellow color indicates nuclear colocalization between STAT3-P^S727 or NF-κB with HMGB1. (C) Detection of STAT3 and NF-κB activation by cellular fractionation. Cytoplasmic (C) and nuclear (N) proteins were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents. Fifty micrograms of proteins was loaded onto each lane. Mouse anti-STAT3-P^S727 or NF-κB antibody was used for determination of nuclear translocation of each protein, and mouse anti-Rb and rabbit anti-LDH antibodies were used as marker for nucleus and cytoplasm, respectively. CD68 was used as a marker for NLCs.