35-9200

antibody from Invitrogen Antibodies

Targeting: PRLR

Provider product page for 35-9200

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [33]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [33]

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Product number: 35-9200 - Provider product page
Provider: Invitrogen Antibodies
Product name: Prolactin Receptor Monoclonal Antibody (1A2B1)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: This antibody is predicted to react with mouse and rat and reacts with the long form of the human prolactin receptor (hPRLr) protein at ~85-90 kDa, and may also identify the human prolactin receptor intermediate and delta S1 isoforms by western blotting. On immunoprecipitation, this antibody strongly recognizes only the long form of hPRLr. During development reactivity was confirmed with human T-47D breast carcinoma cells.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: 1A2B1
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

PRLR protein structure - 35-9200 shown in red.

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Experimental details: Fig 3 Detection of PrlR in human LAM cells using immunofluorescence. Staining LAM/TSC cells using different PrlR antibodies detect a significant immunofluorescence signal in LAM/TSC cells. The antibodies employed were U5 (Thermoscientific) and A12B1 (Invitrogen).

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Experimental details: Figure 3 Expression of pGL4-CISH to PRL and PRLr in different human breast cancer and epithelial cell lines. Cells were transfected with pGL4-CISH. After transfection, cells were arrested in minimal defined medium without FBS, the transfectants were stimulated with PRL for 24 hours prior to analysis of luminescence. A. Cells lines were transfected with 50 ng pGL4-CISH/1 x 10 5 cells. B. MCF7 cells were overexpressed with pEF-PRLr and treated with PRL (200 ng/ml). *, P < 0.05. ***, P < 0.001. C. The low inset is Western blot demonstrating PRLr overexpression level obtained with the various transfectants with the arrows indicating each lane of treatments. Error bars represent SEM; * denotes P < 0.05, and *** denotes P < 0.001 as compared with no PRL stimulation alone.

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Experimental details: Fig. 2 Establishment of T47D/PRL clones with high levels of ectopic PRL expression a ISH analysis of PRL mRNA expression in wild type and PRL-transfected cell lines. Bright field (BF) and dark field (DF) pictures are shown. Sections of paraffin-embedded cells were hybridized with an antisense cRNA probe (507 bp) complementary to the PRL mRNA (AS PRL). Signal is visualised under DF as white grains . BF represents the haematoxylin and eosin stained sections after hybridization with AS PRL probe to show morphology of the cells. Under BF ISH signal is visualised as black grains . Lack of detectable hybridization signal with sense probe (S PRL) indicates specificity of signals obtained with the PRL AS probe. Sections were developed after 2 weeks of exposure. Scale bars = 100 mum. b Analysis of the PRL protein expression. Cells were grown to 70-80 % confluence in 10 % FCS medium and then starved for 24 h in serum-free DMEM medium. 20 mug of cellular protein extracts were analysed by Western blotting using a PRL-specific antibody ( top panel ); beta-actin was used as protein loading control ( lower panel ). Supernatants were analysed for secreted and active PRL using ELISA and Nb2 proliferation, respectively. The PRL level is stated as nmoles/1 mio cells/24 h. b.d. below detection limit. c Transwell co-culture of T47D and T47D/PRL cells. T47D Tet-On cells were seeded in 24-well multi-dishes in 10 % CSS medium. After 24 h of plating, inserts with either T47D Tet-On or T47D/pTRE-PRL C

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Experimental details: Fig. 4 Ectopically expressed PRL down-regulates PRLR expression and responsiveness to exogenous rPRL stimulation a PRLR protein expression in T47D/PRL cells. Cells were harvested at 70-80 % confluence and 40 mug protein extracts were analysed by Western blotting using an anti-human PRLR antibody. beta-actin was used as protein loading control. The signals of PRLR were quantified and normalised by the beta-actin signals. The numbers represent a percentage of the respective control T47D cell line. Expression of PRLR on the surface of the cells was analysed by flow cytometry (FACS). Cells were stained with an anti-human PRLR mAb followed by anti-mouse IgG-APC. The median fluorescent intensities (MFI) are shown. b Cell signalling induced by rPRL. Cells were grown to 70-80 % confluence in 10 % FCS medium and starved in serum-free DMEM medium for 24 h prior to treatment with 20 nM rPRL for 30 min. 20 mug of protein extracts were analysed for activation of the ERK1/2 pathway by Western blotting using an anti-pERK1/2 antibody. Membranes were stripped and sequentially re-probed with antibodies detecting total ERK1/2 protein and beta-actin as loading controls. c Proliferation of T47D/PRL cells upon E2 and PRL exposure. Cells were seeded with a density of 1.2 x 10 4 cells/well in 96-well multi-dishes in 10 % CSS medium. Next day, medium was renewed and cells were cultured for three days in absence or presence of 1 nM 17beta-estradiol (E2) and 20 nM rPRL as indicated. Cell proliferation

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Experimental details: Fig 1 Effect of TSC2 siRNA on GHR and PrlR expression. The mouse cell line CRL-2620 was transfected with two different TSC2 siRNAs (siRNA_1 and siRNA_3), or a control siRNA (si_C). (A) Quantitative RT-PCR was used to test the efficacy with which these siRNAs down-regulated TSC2 mRNA. The most pronounced effect (approximately 60% reduction) was seen with TSC2 siRNA_3, which was used for further experiments. (B) The figure shows a representative Western blot probed with antibodies directed to GHR, PrlR, SOCS3 and GAPDH; iGHR and mGHR, are the immature and mature forms of GHR, respectively. (C) Densitometric analyses of Western blots. Protein lysates were prepared individually from three separate culture dishes treated with TSC2 siRNA or control siRNA and subject to Western blot analysis; experiments were repeated three times and gave consistent results. * = P-value

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Experimental details: Fig 2 Immunofluorescence of CRL-2620 using PrlR and GHR antibodies. Cells were transfected with TSC2 or control siRNAs. After over-night incubation, the cells were washed 3 times with PBS and incubated with antibodies for PrlR and GHR. The figures show that PrlR immunostaining was markedly increased following TSC2 knock-down. A strong immunofluorescence signal was observed at intracellular locations. In the negative control panel, cells were incubated with mouse IgG and rabbit IgG antibodies. Fluorescence images were acquired at a magnification of 63x.

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Experimental details: Fig 3 Detection of PrlR in human LAM cells using immunofluorescence. Staining LAM/TSC cells using different PrlR antibodies detect a significant immunofluorescence signal in LAM/TSC cells. The antibodies employed were U5 (Thermoscientific) and A12B1 (Invitrogen).

Supportive validation

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Experimental details: Fig 4 Prolactin receptor immuno reactivity in human LAM lung lesions. Histological sections from LAM patients were analyzed. Human lung tissues were obtained from subjects who underwent lung resection for diagnostic reasons and immunohistochemical-staining using the PrlR antibody clone 1A2B1 (Invitrogen) was carried out. Arrows indicate the location of PrlR immuno reactivity in one microscope field of a representative image from LAM tissue; compared to positive cells for PrlR in control lung tissue. For negative control, primary antibody was omitted, Scale bars: 50 mum.

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Experimental details: Fig 5 Human LAM cells express PrlR, and prolactin stimulates phosphorylation of STAT3 and Erk. LAM/TSC cells were cultured in serum-free medium overnight. The cells were then exposed to different doses of PrlRA (20, 50, 200 ng/ml) for 15 minutes, and subsequently the cells were exposed to 200 ng/ml Prl for 60 minutes or PBS as a control. Then protein extracts were prepared for Western blot analysis, and probed with antibodies for P-STAT3, STAT3, P-Erk and Erk. (A) Western blot to analyze PrlR in LAM/TSC control cells; an antibody against human PrlR detected a protein band of 89-90 kD. (B) Western blot, using antibodies directed against P-STAT3, STAT3, P-Erk and Erk in LAM/TSC cells following treatment as described in Material and methods section. (C) Densitometric quantification of Western blot signals, in which the Y-axis depicts the ratio between phosphorylated STAT3 and Erk to the total protein.

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Experimental details: Fig 6 Immunohistochemical staining of the prolactin receptor and CD34 in mammary carcinoma. (A) Staining of the prolactin receptor (turquoise) and CD34 (endothelial cells, purple), counterstaining with haematoxylin (blue). The pictures were digitally processed to unmix the different colours. (B) Fluorescence-like image of both prolactin receptor (green) and CD34 (red) in pseudo-colours. (C) Areas where prolactin receptor and CD34 co-localize, and endothelial expression of prolactin receptor. (D) and (E), respectively, show pseudo-fluorescent staining for the prolactin receptor (green), endothelial cells (red) and nuclei (blue). Scale bar = 0.075 mum in (A-E).

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Experimental details: Figure 4 High density/ stiff collagen matrices enhance E2 and PRL induced cell growth, and inhibit apoptosis in T47D cells. a) T47D cells were cultured in LD or HD collagen and treated +- 1nM E2, +- 4nM PRL for 7d. Cells were removed from gels and counted via hemocytometer. n = 4. b) T47D cells were cultured in LD or HD collagen and treated +- 1nM E2, +- 4nM PRL for 72h and lysates were immunoblotted with the indicated antibodies. c) T47D cells were cultured as in (a) , and treated +- 4-OHT 1h prior to hormone treatments and quantitation of cell number. n = 4. d,e,f,g) T47D cells were cultured as in (b) , fixed and stained for DAPI and Ki-67 (d,e) or cleaved caspase 3 (f,g) , and quantified as described in the Materials and Methods. n = 3. Representative images of DAPI/ Ki67 are shown in (e) and DAPI/ cleaved caspase 3 are shown in (g). Different letters represent significant differences (p

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Experimental details: Figure 3 E2 and PRL co-treatment inhibits MCF-7 cell growth and proliferation in low density/ compliant but not high density/ stiff collagen matrices. a) MCF-7 cells were cultured in LD or HD collagen and treated +- E2, +- PRL for 72h. Cells were removed from gels and counted. n = 3. b) MCF-7 cells were cultured and treated as in (a) and lysates were immunoblotted with the indicated antibodies. Representative blots shown. c) MCF-7 cells were cultured as in (a) and treated +- 4-OHT 1h prior to hormone treatment. n = 4. d,e) MCF-7 cells were cultured and treated as in (a) . Gels were fixed and stained for DAPI and Ki-67, and quantified as described in the Materials and Methods (d). n = 3. e) Representative images of DAPI/ Ki-67 are shown in (d). Different letters represent significant differences (p

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Experimental details: Fig. 1 MCF10AT hPRLrL + I co-expression in vivo. a MCF10AT cells were stably transfected with either hPRLrL, hPRLrI, both isoforms together (at a 1:1 ratio), or empty vector (MCF10AT-hPRLrL, MCF10AT-hPRLrI, MCF10AT-hPRLrL+I, and MCF10AT-EV, respectively), and expression was confirmed by IB. b These cells were orthotopically xenografted into female NSG mice, and primary tumor growth was monitored longitudinally c Lungs were harvested and micrometastatic burden was counted manually. Representative images (scale bar, 100 um) of H&E stained d primary tumor, e axillary lymph node and f lung indicate primary tumors to be high grade adenocarcinoma with nodal infiltration and successful distant metastatic dissemination (outlined; representative image shows metastatic growth long a blood vessel). * p < 0.05, ** p < 0.01, *** p < 0.005. n = 4 mice/study arm. Data is shown as mean +- SEM.

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Experimental details: Fig. 6. Decreased STAT5 activation in Rab6a -deficient mammary glands and RAB6A-depleted T47-D cells. (A) Western blots for pSTAT5, total STAT5 and actin performed on three distinct control and mutant mammary gland protein extracts at P18 and L1 (left). Quantification of pSTAT5/STAT5 amounts (mean+-s.e.m.) (right). * P =0.038 (P18), * P =0.041 (L1). (B) Western blots for pSTAT5, total STAT5, RAB6 and actin performed with T47-D cell lysates (left). SiCtrl and SiRAB6A cells were stimulated with PRL for 5, 15 and 45 min. Quantification of pSTAT5/STAT5 amount at each time point (right). Data are mean+-s.e.m. of four separate siRNA assays. *0.01< P