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. 2011 Aug;31(16):3208–3222. doi: 10.1128/MCB.05337-11

Fig. 1.

Fig. 1.

(A to C) In vitro protein complex formation of ERα with SP1 and C/EBPβ. (D) Inhibition of E2-induced PRLR mRNA by knockdown of SP1, C/EBPβ, and ERα. (A) Western blot of individual, simultaneous, or sequential incubation of in vitro-translated V5-tagged human SP1 and/or human C/EBPβ with GST-ERα (human; aa 185 to 595 with the deletion of the AF1 domain) in the absence or presence of E2 (100 nM). Results are representative of three independent experiments. (B) Dose-dependent effect of SP1 on C/EBPβ or of C/EBPβ on SP1 association with GST-ERα in the simultaneous incubation study. Increasing doses of TnT reticulocyte extracts (top, 5 to 40 μl for SP1-V5 with constant 10 μl C/EBPβ; bottom, 10 to 40 μl of C/EBPβ with constant 10 μl SP1) were incubated with GST-ERα. Following pulldown assays (see Materials and Methods) for the detection of SP1-V5 or C/EBPβ-V5 association with ERα, V5 antibody was used in the Western blot analysis. *, SP1 protein variant translated from an alternative ATG codon. (C) Protein-protein interaction was independent of the DNA template used for in vitro transcription/translation. The upper gel shows human ERα-V5 plasmid (1 μg), used for the TnT coupled reticulocyte lysate system, treated with DNase (100 U, 1 h, 25°C) and resolved in a 1% agarose gel. Five hundred thirty-eight bp of ERα-V5 was amplified by PCR with a primer (+1/537 bp; NM_000125). The lower gel shows 10 μl of TnT reticulocyte extract used in the study for the Western blotting of ERα-V5 pulldown by GST-SP1. (D) Effect of E2 on the expression of hPRLR mRNA in MCF7 cells with the knockdown of transcription factors (individually and combined). Cells were transfected with SP1 or C/EBPβ siRNA singly or combined or with ERα siRNA or scramble siRNA (control). After 48 h of transfection, cells were left untreated or were treated with 17β-estradiol (100 nM) for 24 h. hPRLR mRNA expression was determined by real-time PCR analyses of hPRLR transcripts from total RNA. The inset shows a Western blot of endogenous SP1, C/EBPβ, or ERα in cell lysates after siRNA treatment. Results are normalized to results for β-actin and are means ± standard errors of duplicate experiments in triplicate.