Figure 6.

Sp1 and Sp3 distribution relative to ERα. (A) Seven A₂₆₀ of lysate from MCF-7 cells grown in complete medium were incubated with 5 μg of anti-Sp1 or anti-Sp3 antibodies or with normal rabbit IgG nonspecific antibodies as negative control, and the immunoprecipitated (IP, lanes 2, 4, and 6) and immunodepleted (ID, lanes 3, 5, and 7) fractions were collected. The whole IP fractions and equivalent volumes of lysate (lane 1) and ID fractions, corresponding to 1% of 7 A₂₆₀ of lysate were loaded onto a SDS-10% polyacryl-amide gel, transferred to nitrocellulose membranes, and immunochemically stained with anti-ERα antibodies. (B) Nine A₂₆₀ of lysate from MCF-7 cells grown in complete medium were incubated with 4 μg of anti-ERα or antiintegrin (control) antibodies, and the immunoprecipitated (IP, lanes 2 and 4) and immunodepleted (ID, lanes 3 and 5) fractions were collected. The whole IP fractions and equivalent volumes of lysate (lane 1) and ID fractions, corresponding to 0.5% of 9 A₂₆₀ of lysate were loaded onto a SDS-10% polyacryl-amide gel, transferred to a nitrocellulose membrane, and immunochemically stained with anti-Sp1 antibodies. After stripping of the anti-Sp1 antibodies, the membrane was restained with anti-Sp3 antibodies. (C and D) MCF-7 cells were grown on coverslips in complete medium, fixed, and double labeled with anti-Sp1 or anti-Sp3 and anti-ERα antibodies. Spatial distribution was visualized by fluorescence microscopy and image deconvolution as described in Materials and Methods. Single optical sections are shown. Yellow in the merged images signifies colocalization. The merged images were analyzed by the generation of a horizontal linescan (top box) and a diagonal linescan (bottom box) along with a correlation scatter plot. Bar, 5 μm.