The results of these analyzes show a significant increase in PRL mRNA expression at 48 h and 72 h compared to non-transfected MCF-7 cells, but this was not the case after 24 hours. 1D shows the Western blot of pit 1, PRL, actin and b in MCF-7 cells with pRSV HPIT 1 or pRc / RSV transfected. Pit 1 has two major immunoreactive bands that are easily seen in samples from human cell lines and mammary AS-605240 glands. The resulting B Santander from two other translation initiation codons in the Pit 1 mRNA were been called on 31 and 33 kDa bands. PRL also shows two immunoreactive bands that are not glycosylated and glycosylated forms. As expected, transfection of MCF-7 cells with a construct pRSVhPit a significant increase in protein expression of Pit 1 induced. PRL was detectable in control cells MCF 7, and the PRC / RSV-transfected cells.
H Was here PRL expression observed after 48 h in a pRSV HPIT construct pRC / RSV-transfected cells compared. In order to assess whether the pit a knockdown Ver Induced changes in PRL siRNA used. MCF-7 cells were transfected with siRNA missense or Pit 1 siRNA. AZD8055 As shown in Fig. 1E, transfection of MCF-7 cells with 20 nM siRNA missense not Pit 1 or PRL expression compared to transfected cells from controlled change about. However, knockdown of Pit 1 through transfection with 20 nM siRNA Pit 1 at a significantly reduced expression of both PRL and Pit-1. MCF-7 cells containing only PRL proximal promoter transcripts pituitary PRL mRNA was demonstrated in extrapituitary tissues, that of the hypophys Material from other sources, suggesting different mechanisms of gene regulation by PRL.
Thus, using RT-PCR, we kind of PRL mRNA transcribed in the cell line model experimental system was evaluated. As shown in Fig. 2B is the human pituitary tissue, NIH 3T3 cells, MCF-7 cells and HeLa cells were obtained by RT-PCR using primers that are specific transcripts PRL extrapituitary and the pituitary. Expressing W While HeLa cells, both the pituitary and extrapituitary PRL mRNA transcripts showed the MCF-7 cells, a single band of 194 bp, as seen in the PCR of the human pituitary gland extract. Pit 1 regulates the expression of PRL in MCF-7 cells by binding to the PRL proximal promoter To the m Possible effect of a pit on the transcriptional activity of t judge of PRL were MCF-7 cells either with a construct linking transfected 217 bp simply PRL proximal promoter of the gene to a luciferase reporter vector or the vector pGL2, then with a vector pRSV HPIT co-transfected.
after 48 h after transfection, the cells for the measurement Luciferaseaktivit t were harvested. As shown in Fig. 2D, cotransfection with pRSV HPIT hPRLK216/C1 pGL2B and 1 leads to a significant increase in the Luciferaseaktivit t. To specify the location of the tomb of a PRL binding to the promoter, we performed site-directed mutagenesis of two known Pit 1 binding sites and then cotransfected these constructs with a vector pRSV HPIT expression. As shown in Fig. 2D is the increase in the transcription using the wild type pGL2B hPRLK216/C1 construction was significantly reduced when the mutated promoter constructs with the PRL HPIT a pRSV vector were cotransfected, indicating that these elements. For the best in vivo interaction of the pit a Wi Term