Figure 3

Post-translational regulation of GATA3 through the 26S proteasome. A) T47D-Y-C587A cells were treated with MPA for 18 hours, protein cell lysates were prepared and immunoblotted for the indicated proteins. Signal intensities of GATA3 bands in the WBs were analyzed by densitometry, values were normalized to GAPDH protein bands setting the value of untreated cells as 1.0. B) T47D cells were treated or not with MPA and/or actinomycin D (5 μg/μl) for the indicated times. The fold change of mRNA expression levels upon MPA treatment for the indicated times was calculated by normalizing the absolute levels of GATA3 mRNA to GAPDH levels, which were used as an internal control, and setting the value of untreated cells as 1.0. Not significant (NS), one-way ANOVA. C) T47D cells were treated or not with cycloheximide (CHX) at 1 μM final concentration and/or with MPA or bortezomib (Btz) at 100 nM final concentration as indicated, protein cell lysates were prepared and GATA3 expression was measured by WB. Signal intensities of GATA3 bands in the WBs were analyzed as indicated in A. D) T47D cells were pretreated or not with Btz for 30 minutes and then treated or not with MPA as indicated, and GATA3 mRNA expression was measured by RT-qPCR as described for B (***P <0.001, one-way ANOVA). E) T47D cells were treated or not with MPA and/or Btz as indicated, protein cell lysates were prepared and GATA3 expression was measured by WB. Signal intensities of GATA3 bands in the WBs were analyzed as indicated in A. ANOVA, analysis of variance; MPA, medroxyprogesterone acetate; WB, Western blot.