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. 2010 Sep 27;30(23):5456–5472. doi: 10.1128/MCB.00012-10

FIG. 1.

FIG. 1.

MPA effects on ErbB-2 and Stat3 activation and cellular localization. (A) MPA induces rapid ErbB-2 phosphorylation via the classical PR. Cells were treated with MPA or pretreated with RU486 and transfected with PR or control siRNAs before MPA stimulation. Western blots (WB) were performed with pErbB-2 antibodies, and filters were reprobed with a total ErbB-2 antibody. The Western blot of C4HD cells at the bottom shows the effects of siRNAs on PR expression. (B) c-Src mediates MPA-induced ErbB-2 activation. Cells were treated with MPA or preincubated with PP2 before MPA treatment. Western blots were performed with phosphoprotein antibodies, and membranes were reprobed with total protein antibodies. (C) Rapid progestin effects mediate the activation of ErbB-2. T47D-Y cells were transfected with either the PR-BmPro or C587A-PR mutant and were then treated with MPA. Western blots were performed with pErbB-2 antibodies, and filters were reprobed with total ErbB-2 antibody. (D) MPA induces ErbB-2 nuclear migration. (Top) Cells were treated with MPA for the time points shown, and nuclear and cytosolic protein extracts were analyzed by Western blotting. The pTyr 1272/1222 ErbB-2 blot was reprobed with the ErbB-2 carboxy-terminal region antibody, and the pTyr 927/877 blot was reprobed with the antibody to the ErbB-2 amino terminus. Total cell lysates were blotted in parallel. ErbB-2 in untreated cells remained in the cytoplasmic membrane, which was not analyzed in this Western blot (see Fig. 2A for an image of ErbB-2 membrane localization in cells not treated with MPA). (Bottom) Western blot showing that the inhibition of ErbB-2 phosphorylation with AG825 blocks ErbB-2 nuclear migration. Histone H3 and β-tubulin were used to control cellular fractionation efficiency. (E) MPA induces Stat3 activation via ErbB-2. Cells were treated with MPA or pretreated with AG825. C4HD cells were also transfected with ErbB-2 siRNAs targeting mouse ErbB-2 and with control siRNAs. Western blots were performed with phospho-antibodies, and filters were reprobed with the respective total protein antibody. (F) MPA stimulates Stat3 nuclear translocation. Nuclear and cytosolic protein extracts were analyzed by Western blotting with pStat3 antibody. Blots were reprobed with total Stat3 antibody. The experiments for which the results are shown were repeated five times, with similar results.