Fig. 1

STAT3 and HP1α physically interact. (a) A549 cells were fixed and immunostained with anti-STAT3 (Green) and anti-HP1α (Red) and were imaged using confocal microscopy. A 1 μm optical section is shown to reveal colocalization in discrete regions (arrows). (b) A549 cell lysates were immunoprecipitated with anti-STAT3 (top panel) or anti-HP1α (lower panel) antibodies, the presence of HP1α and STAT3 in the immunoprecipitates were detected by Western blotting with the respective antibodies. (c, d) A549 cells were fixed and immunostained with anti-HP1α-Alexa488 (donor) to anti-STAT3-Alexa546 (acceptor). Donor and acceptor bleed through was corrected using donor and acceptor only samples. FRET was detected and processed using a Leica confocal microscope with built-in FRET software. Note that FRET is detected in the nucleus (C), that IL6 treatment reduced FRET efficiency (d). (e) FRET efficiency was quantified as YFP/CFP fluorescence ratio for cells with or without IL-6 treatment. ** indicates p < 0.01 in unpaired Student’s t-test. Scale bars, 2 μm