FIG. 4.

MPA induces association of Stat3 with PR. (A) C4HD cells were treated with 10 nM MPA for the indicated times or preincubated with PP2 before MPA treatment for 5 min, and PR was immunoprecipitated from 500 μg of protein extracts. As a control, lysates were also immunoprecipitated with normal mouse serum (NMS). Immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting with an anti-Stat3 antibody (upper part). Twenty micrograms of protein from cell extracts was directly immunoblotted with the Stat3 antibody (last lane, upper part). Identical aliquots of each immunoprecipitate were subjected to immunoblot analysis with anti-PR antibody to verify that nearly equal amounts of immunoprecipitated proteins were loaded (lower part). (B) Protein lysates (500 μg) from cells treated as indicated in panel A were immunoprecipitated with an anti-Stat3 antibody or with normal rabbit serum (NRS). Immunocomplexes were subjected to SDS-PAGE and analyzed by Western blotting with an anti-PR antibody (upper part). Twenty micrograms of protein from cell extracts was directly immunoblotted with the PR antibody (last lane, upper part). Identical aliquots of each immunoprecipitate were subjected to immunoblot analysis with anti-Stat3 antibody to verify that nearly equal amounts of immunoprecipitated proteins were loaded (lower part). This is a representative experiment out of a total of three. W, Western blot assay; IP, immunoprecipitation.