Journal: International journal of oncology

Article Title: The prolactin receptor mediates HOXA1-stimulated oncogenicity in mammary carcinoma cells.

doi: 10.3892/ijo.2012.1660

Figure Lengend Snippet: Figure 3. Forced expression of HOXA1 enhanced PRL-stimulated MAPK activation in human mammary carcinoma cells. (A) Western blot analysis. Cells were serum deprived in serum‑free media for 24 h, followed by 15 min stimulation with 2 µg/ml of hPRL. Soluble whole cellular extracts were run on a 12.5% SDS-PAGE and immunoblotted using an anti-phospho-p44/42 MAPK antibody (Cell Signalling Technology) for phosphorylated p44/42 protein and an anti-p44/42 MAPK antibody (Santa Cruz Biotechnology) for total p44/42 protein as loading control for cell lysates. The sizes of detected protein bands in kDa are shown on the right. (B) ERK reporter assay. ERK1/2 (p44/42)‑depen dent transcription was measured by using an ERK luciferase reporter gene plasmid, which contains an egr-1 promoter fragment comprising 624 bp 5' of the initiation codon. MCF7-HOXA1 (solid bar) and MCF7‑VECTOR (open bar) cells were co-transfected with the reporter plasmid together with a β-galactosidase reporter vector as the control for transfection efficiency in serum-free RPMI with (+) or without (-) 2 µg/ml of recombinant human pro lactin (hPRL). After a further 24 h, luciferase and β-galactosidase activities were quantified using a kit (Promega). Luciferase activity values were normal ized to β-galactosidase activity and presented as mean ± SE (standard error of the mean) of three independent experiments each in triplicate and are pre sented relative to the mean of MCF7-VECTOR cells without hPRL treatment. The effect of hPRL treatment is shown as fold increase in comparison with the untreated control. *P<0.05; **P<0.01.

Article Snippet: Western blot analysis was essentially performed as previously described (45) using the following antibodies: anti-PRLR monoclonal antibody U5 (Abcam, Cambridge, UK); antiphospho-STAT5A/B antibody (Millipore, Billerica, MA); anti-Stat5 and anti-p44/42 MAPK antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phospho-p44/42 MAPK antibody (Cell Signaling Technology, Beverly, MA); anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO).

Techniques: Expressing, Activation Assay, Western Blot, SDS Page, Control, Reporter Assay, Luciferase, Plasmid Preparation, Transfection, Recombinant, Activity Assay, Comparison