Fig. 7

ERα-36 recruits p300 and mediates IL-6 induced STAT3 acetylation. a and b, quiescent MDA-MB-231 cells were treated with and without IL-6 for the indicated time periods, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were immunoprecipitated (IP) with anti-ERα-36 or anti-p300 antibodies, and the immunocomplexes were analyzed by Western blotting using anti-p300 and/or anti-ERα-36 antibodies. IB, immunoblot. c, quiescent MDA-MB-231 cells were treated with and without IL-6 in the presence and absence of TAM (10 μM) for 1 h, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were immunoprecipitated with anti-ERα-36 antibodies, and the immunocomplexes were analyzed by Western blotting using anti-p300 antibodies. d, all the conditions were the same as in B except that MDA-MB-231 cells were transduced with Ad-GFP or Ad-dnERα-36 and quiesced before being treated with IL-6. Using with anti-ERα-36 antibodies to show equal amounts of immunoprecipitated target protein. e, f and g, MDA-MB-231 cells that were transfected with scrambled (Scr) or p300 siRNA (100 nM) and quiesced were treated with and without IL-6 for 1 h, and cell extracts were prepared. Cell extracts containing an equal amount of protein from control and each treatment were analyzed by Western blotting using anti-pSTAT-3 and anti-acetyl-STAT3 antibodies (e) using anti-MMP2 and anti-MMP9 (g) antibodies or subjected to immunoprecipitation using anti-ERα antibodies followed by immunoblotting using anti-acetyl-STAT3 antibodies (f). The blots were reprobed sequentially with anti-pSTAT3, anti-STAT3, and anti-ERα-36 antibodies. h, MDA-MB-231 cells that were transfected with scrambled or p300 siRNA (100 nM) in combination with either empty vector or pGL3-MMP2-luc and pGL3-MMP9-luc, and cell extracts were prepared and analyzed for luciferase activity. RLU, relative luciferase units. i, MDA-MB-231 cells that were transfected with scrambled (Scr) or p300 siRNA (100 nM) and quiesced were treated with and without IL-6 for 1 h induced MDA-MB-231 cell migration. Bar graphs represent means ±S.D. values of three independent experiments. (**, P<0.01, ^#, P>0.05) n = 3