monoclonal prlr-specific antibodies  (Novartis)

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: Mutation of the prolactin receptor transactivation domain prevents its phosphorylation and impairs global PRL-induced gene expression. A: Western blot analysis demonstrating PRLr or PRLrYDmut overexpression in MCF7 stable cell lines using an anti-PRLr or an anti-V5 antibody (to detect only epitope-tagged PRLr). Comparison to endogenous PRLr in T47D cells demonstrates that stable cell lines express physiological levels of PRLr. B: Mutation of the PRLr TAD (PRLrYDmut) impairs phosphorylation of Y406. MCF7 cells were serum starved, treated with PRL, lysed, and subjected to immunoprecipitation (IP) analysis using an anti-V5 antibody. Eluted samples were analyzed by using Western blot analysis with an anti–phospho-Y406 PRLr antibody. Blots were stripped and reprobed for anti-V5 to show proper pull down. C: Heat maps of genes identified in microarray analysis were generated with unsupervised hierarchical clustering. MCF7 stable cells were serum starved for 24 hours, then treated with PRL for 2 hours. Data reveal a global view of PRL-induced genes up-regulated in MCF7 PRLr-expressing cells compared with MCF7 transactivation-deficient PRLrYDmut-expressing cells. D: Heat map of genes differentially regulated by PRL. In C and D, the biological replicates of WT, WT + PRL, and MUT + PRL group together, whereas replicates of MUT are grouped with empty control samples. Labels specify samples/treatments (empty, empty vector; WT, PRLr; MUT, PRLrYDmut; +P, PRL treatment) and biological repeats (ie, empty.A, empty.B, and empty.C). The scale is provided, where green represents down-regulated genes and red represents up-regulated genes. E: Venn diagram of PRL-induced genes up-regulated by PRLr or PRLrYDmut. Gene lists comparing WT + PRL versus empty + PRL or MUT + PRL versus empty + PRL (1.2-fold up-regulation, P < 0.05) were generated. F: Venn diagram of transcripts regulated by PRLr expression or PRL treatment. Gene lists comparing PRLr versus empty or empty + PRL versus empty (1.2-fold up-regulation, P < 0.05) were generated. The results demonstrate many genes regulated by PRLr expression and PRL treatment and 486 genes that are shared between both groups. G and H: Real-time PCR demonstrates that IER3 (G) and CCND1 (H) are up-regulated in a PRL-induced manner by expression of PRLr, but not PRLrYDmut. Ctrl, control; mut, prolactin receptor harboring Y406F/D411A mutations; PY406, phosphorylation on PRLr residue tyrosine 406. ***P < 0.001.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques: Mutagenesis, Expressing, Western Blot, Over Expression, Stable Transfection, Immunoprecipitation, Microarray, Generated, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: PRLr Y406 phosphorylation is increased as a function of neoplastic progression. A–C: Data analysis performed using the Oncomine database. Representative graphs demonstrating that the PRLr is overexpressed in ductal breast cancer compared with normal breast tissue (A),56 the PRLr is more highly expressed in ER+ versus ER− breast tumors (B),59 and the PRLr is more highly overexpressed in PR+ versus PR− breast tumors (C).61D: Representative anti–phospho-Y406 PRLr IHC from patient-matched normal, primary tumor, and lymph node metastasis breast tissue. The photomicrograph demonstrates the observed increase in PRLr Y406 phosphorylation in malignant tissue, with significant accumulation in the cell nucleus. Original magnification, ×400. E: Quantification of nuclear PRLr Y406 phosphorylation. F: Quantification of cytoplasmic PRLr Y406 phosphorylation. The results are shown as the mean ± SEM, and P values were determined using a one-way analysis of variance. G: Values from TMA analysis were stratified into ER− and ER+ groups for analysis, and a two-way analysis of variance was performed to analyze the effects of both ER status and tumor status. Statistical analysis demonstrated a significant increase in Y406 phosphorylation as a function of tumor status and ER positivity, although no individual groups were significantly different from each other, as analyzed by the post hoc Bonferroni test. Normal, normal breast tissue; Tumor, primary breast tumor; LN Met, lymph node metastasis. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques:

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: Correlation between ERα, PR, and PRLr expression results from a tripartite regulatory loop. Model demonstrating the relationship between PRLr, ERα, and PR. Herein, perturbation of the PRLr, PRLr Y406 phosphorylation, ERα, or PR level disrupts the balance in expression of all three receptors.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques: Expressing

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: Mutation of the prolactin receptor transactivation domain prevents its phosphorylation and impairs global PRL-induced gene expression. A: Western blot analysis demonstrating PRLr or PRLrYDmut overexpression in MCF7 stable cell lines using an anti-PRLr or an anti-V5 antibody (to detect only epitope-tagged PRLr). Comparison to endogenous PRLr in T47D cells demonstrates that stable cell lines express physiological levels of PRLr. B: Mutation of the PRLr TAD (PRLrYDmut) impairs phosphorylation of Y406. MCF7 cells were serum starved, treated with PRL, lysed, and subjected to immunoprecipitation (IP) analysis using an anti-V5 antibody. Eluted samples were analyzed by using Western blot analysis with an anti–phospho-Y406 PRLr antibody. Blots were stripped and reprobed for anti-V5 to show proper pull down. C: Heat maps of genes identified in microarray analysis were generated with unsupervised hierarchical clustering. MCF7 stable cells were serum starved for 24 hours, then treated with PRL for 2 hours. Data reveal a global view of PRL-induced genes up-regulated in MCF7 PRLr-expressing cells compared with MCF7 transactivation-deficient PRLrYDmut-expressing cells. D: Heat map of genes differentially regulated by PRL. In C and D, the biological replicates of WT, WT + PRL, and MUT + PRL group together, whereas replicates of MUT are grouped with empty control samples. Labels specify samples/treatments (empty, empty vector; WT, PRLr; MUT, PRLrYDmut; +P, PRL treatment) and biological repeats (ie, empty.A, empty.B, and empty.C). The scale is provided, where green represents down-regulated genes and red represents up-regulated genes. E: Venn diagram of PRL-induced genes up-regulated by PRLr or PRLrYDmut. Gene lists comparing WT + PRL versus empty + PRL or MUT + PRL versus empty + PRL (1.2-fold up-regulation, P < 0.05) were generated. F: Venn diagram of transcripts regulated by PRLr expression or PRL treatment. Gene lists comparing PRLr versus empty or empty + PRL versus empty (1.2-fold up-regulation, P < 0.05) were generated. The results demonstrate many genes regulated by PRLr expression and PRL treatment and 486 genes that are shared between both groups. G and H: Real-time PCR demonstrates that IER3 (G) and CCND1 (H) are up-regulated in a PRL-induced manner by expression of PRLr, but not PRLrYDmut. Ctrl, control; mut, prolactin receptor harboring Y406F/D411A mutations; PY406, phosphorylation on PRLr residue tyrosine 406. ***P < 0.001.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques: Mutagenesis, Expressing, Western Blot, Over Expression, Stable Transfection, Immunoprecipitation, Microarray, Generated, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: PRLr Y406 phosphorylation is increased as a function of neoplastic progression. A–C: Data analysis performed using the Oncomine database. Representative graphs demonstrating that the PRLr is overexpressed in ductal breast cancer compared with normal breast tissue (A),56 the PRLr is more highly expressed in ER+ versus ER− breast tumors (B),59 and the PRLr is more highly overexpressed in PR+ versus PR− breast tumors (C).61D: Representative anti–phospho-Y406 PRLr IHC from patient-matched normal, primary tumor, and lymph node metastasis breast tissue. The photomicrograph demonstrates the observed increase in PRLr Y406 phosphorylation in malignant tissue, with significant accumulation in the cell nucleus. Original magnification, ×400. E: Quantification of nuclear PRLr Y406 phosphorylation. F: Quantification of cytoplasmic PRLr Y406 phosphorylation. The results are shown as the mean ± SEM, and P values were determined using a one-way analysis of variance. G: Values from TMA analysis were stratified into ER− and ER+ groups for analysis, and a two-way analysis of variance was performed to analyze the effects of both ER status and tumor status. Statistical analysis demonstrated a significant increase in Y406 phosphorylation as a function of tumor status and ER positivity, although no individual groups were significantly different from each other, as analyzed by the post hoc Bonferroni test. Normal, normal breast tissue; Tumor, primary breast tumor; LN Met, lymph node metastasis. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques:

Journal: The American Journal of Pathology

Article Title: The Prolactin Receptor Transactivation Domain Is Associated with Steroid Hormone Receptor Expression and Malignant Progression of Breast Cancer

doi: 10.1016/j.ajpath.2012.09.021

Figure Lengend Snippet: Correlation between ERα, PR, and PRLr expression results from a tripartite regulatory loop. Model demonstrating the relationship between PRLr, ERα, and PR. Herein, perturbation of the PRLr, PRLr Y406 phosphorylation, ERα, or PR level disrupts the balance in expression of all three receptors.

Article Snippet: The slides were blocked in the blocking buffer (2.5% bovine serum albumin and 0.1% Triton X-100) for 10 minutes and incubated with a phospho-Y406 PRLr-specific antibody (New England Peptide, Ipswich) (1:10 dilution for immunofluorescence; 1:20 dilution for IHC) overnight at 4°C.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

doi: 10.1186/1471-2407-10-678

Figure Lengend Snippet: Characterization of PRLR isoform antibodies. A. Western blot analysis indicating specificity of polyclonal antibodies. CHO cells were transiently transfected with isoform specific PRLR cDNA as described. Cell lysates were prepared and proteins separated by PAGE. Each isoform specific lysate was probed with each isoform specific antibody. Each transfection was performed in triplicate and Western blot analysis performed twice. Data shown are a representative autoradiogram. Molecular weights are marked as indicated: LF = 93 kDa, SF1a = 57 kDa, SF1b = 38 kDa. Cell lysates from CHO transfected cells were immunoprecipitated with their respective antibodies, then immunoblotted with the same antibodies. Each transfection was performed in triplicate and Western blot analysis performed twice. Data shown are a representative autoradiogram. IP indicates immunoprecipitation. B. Western blot analysis using commercially available PRLR antibody. ECD = antibody recognizing extracellular domain C. Fluorescent immunocytochemical analysis. CHO cells were transiently transfected with isoform specific PRLR cDNA as described. Specific staining was observed using isoform specific polyclonal antibodies. The negative control (not shown) lacks primary antibody and appears black. Data shown are representative of triplicate experiments. Magnification 20×.

Article Snippet: After blocking in 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific antibodies (10 μg/ml) for 2 hr at room temperature.

Techniques: Western Blot, Transfection, Immunoprecipitation, Staining, Negative Control

Journal: BMC Cancer

Article Title: Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

doi: 10.1186/1471-2407-10-678

Figure Lengend Snippet: Immunoanalysis on human xenografts. A. Fluorescent immunohistochemistry was performed on MCF7, MDA-MB-231, or T47D cells which were injected into the cleared fat pads of nude mice; once tumors reached 1 cm 2 (measured in two dimensions), they were excised, formalin fixed, sectioned, and stained with the indicated PRLR isoform specific antibodies. B. Immunohistochemical analysis using DAB as chromogen of MCF7, MDA-MB-231, or T47D xenograft tumors. neg = no primary antibody as negative control; DAPI stain to visualize the presence of cells. Photographs are representative from 5 different tumors for each cell line. Magnification 40×.

Article Snippet: After blocking in 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific antibodies (10 μg/ml) for 2 hr at room temperature.

Techniques: Immunohistochemistry, Injection, Staining, Immunohistochemical staining, Negative Control

Journal: BMC Cancer

Article Title: Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

doi: 10.1186/1471-2407-10-678

Figure Lengend Snippet: Message for PRLR isoforms in breast carcinomas. RNA was extracted from 7 ductal (left) and 7 lobular (right) carcinomas and qPCR was performed. Data shown are the mean +/- SE of delta delta Ct values relative to SF1a expression.

Article Snippet: After blocking in 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific antibodies (10 μg/ml) for 2 hr at room temperature.

Techniques: Expressing

Journal: BMC Cancer

Article Title: Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

doi: 10.1186/1471-2407-10-678

Figure Lengend Snippet: Immunohistochemical analysis from ductal breast tissue arrays. Representative photographs taken of ductal carcinoma specimens to demonstrate absent, low, medium, or high amounts of fluorescent red intensity staining. 144 individual ductal specimens were stained with PRLR isoform specific antibodies and analyzed. Scale bar indicates range of red scoring.

Article Snippet: After blocking in 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific antibodies (10 μg/ml) for 2 hr at room temperature.

Techniques: Immunohistochemical staining, Staining

Journal: BMC Cancer

Article Title: Characterization of ductal and lobular breast carcinomas using novel prolactin receptor isoform specific antibodies

doi: 10.1186/1471-2407-10-678

Figure Lengend Snippet: Immunohistochemical analysis from lobular breast tissue arrays. Representative photographs of lobular carcinoma specimens to demonstrate absent, low, medium, or high amounts of fluorescent red intensity staining. 104 individual lobular specimens were stained with PRLR isoform specific antibodies and analyzed. Scale bar indicates range of red scoring.

Article Snippet: After blocking in 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific antibodies (10 μg/ml) for 2 hr at room temperature.

Techniques: Immunohistochemical staining, Staining