Product Image Gallery
Immunofluorescence analysis of Metadherin was done on 70% confluent log phase U2OS cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Metadherin Rabbit Polyclonal Antibody (Product # 40-6400) at 1.5 µg/mL and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.
Immunohistochemistry analysis of Metadherin (C-Term) showing staining in the cytoplasm of paraffin-embedded human skeletal muscle (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Metadherin (C-Term) Rabbit Polyclonal Antibody (Product # 40-6400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.
Western blot analysis of Metadherin was performed by loading 20 µg of PC-3 (lane1), Caco-2 (lane2), K562 (lane3), MDA-MD-468 (lane4), MDA-MD-231 (lane5), Daudi (lane6), SH-SY5Y (lane7) and U-87 MG (lane8) cell lysate using Novex®NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk at 4°C overnight. Metadherin was detected at ~ 75 kDa using Metadherin Rabbit Polyclonal Antibody (Product # 40-6400) at 1-2 µg/mL in 2.5 % skim milk for 3 hours at room temperature on a rocking platform. Goat Anti-Rabbit IgG - HRP Secondary Antibody (G21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
Knockdown of Metadherin was achieved by transfecting PC-3 cells with Metadherin specific siRNAs (Silencer® select Product # s40866, s40864). Western blot analysis (Fig a) was performed using whole cell extracts from the Metadherin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Metadherin Polyclonal Antibody (Product # 40-6400, 2 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Metadherin.
Western blot analysis of (A) MDA-MB-231, (B) MDA-MB-468, (C) TF-1, (D) K562, (E) Caco-2, and (F) PC-3 cell lysates, and (G) mouse testis homogenates using Zymed Rb anti-Metadherin (C-term) (Product # 40-6400).
Antibody specificity was demonstrated by siRNA mediated knockdown of target protein. PC-3 cells were transfected with Metadherin siRNA and loss of signal was observed in Western Blot using Anti-Metadherin Polyclonal Antibody (Product # 40-6400).
Figure 1 Expression of astrocyte-elevated gene-1 and thymidylate synthase in several non-small cell lung cancer cell lines (A) Immunohistochemical staining revealed upregulation of astrocyte-elevated gene-1 (AEG-1) and thymidylate synthase (TS) protein expression in the primary non-small cell lung cancer (NSCLC) cell lines H157, CL1-0, CL1-5, H520, and H292. (B) Expression of AEG-1 and TS proteins in NSCLC cell lines was analyzed with western blotting. (C) AEG-1 and TS gene expression levels were determined by qPCR. *: compare with PC-9, p < 0.05. (D) Pemetrexed concentration-cell viability relationship was determined in eight NSCLC cell-lines. Lower pemetrexed IC 50 values were noted in NSCLC cell lines expressing lower TS levels. (E) Flow cytometry dot plots and quantitative analysis showed apoptosis rates in three representative cell lines (H520, CL1-0, and PC-9) with different TS expression levels. Measurements from control cells and cells treated with pemetrexed are shown.* p < 0.05.
Figure 5 Increased expression of astrocyte-elevated gene-1 and thymidylate synthase after acquired pemetrexed resistance in lung adenocarcinoma (A) Immunohistochemical staining for thymidylate synthase (TS) and astrocyte-elevated gene-1 (AEG-1) proteins in tissue samples from two patients before pemetrexed treatment and after the development of pemetrexed resistance. (B) Immunohistochemical staining-derived H-scores for TS (B) and AEG-1 (C) expression levels were obtained in tissues from six patients before chemotherapy and at disease progression. Mean TS H-score increased from 44.2 at baseline to 104.2 at disease progression ( P = 0.024), which was concomitant with a comparable increase of AEG-1 H-score from 115.0 to 163.0 ( P < 0.001).
Figure 6 MiR-630 suppresses metastasis in a xenograft mouse model A. IVIS luciferase in vivo images of lung metastasis. B. Representative lung metastasis burden of xenografted animals on day 0, 14, 21, 28, 35, 42, 49 after injected with 231-LUC-NC cells or 231-LUC-miR-630 cells.; lung metastasis models were established ( n = 6 per group) as described in ""Materials and methods"" and lung metastasis burden of xenografted animals were monitored using bioluminescent imaging (BLI). C. Lung tissue weight of the mice sacrificed on the day 49. D, E. Representative H and E staining (D) and MTDH immunohistochemical staining (E) of lung sections were performed on day 49 after cell injection. Scale bar, 50 mum. Lung tissues were fixed with 4% paraformaldehyde immediately after isolated for standard immunohistochemistry analysis as standard immunohistochemistry protocol. F. The IRS scores of MTDH expression in lung sections of mice orthotopically injected with 231-LUC-NC or 231-LUC-miR-630 cells. Data represent mean +- SD. * P < 0.05; ** P < 0.01; *** P < 0.001.
Figure 5 MTDH partially mediates pathology functions of miR-630 in breast cancer A. Western blot analyzing the expression of MTDH in 231-LUC cells and BT-549 cells transfected with miR-NC or miR-630 mimics together with either pcDNA3.1-MTDH or control vector with beta-actin as a loading control. B. Transwell migration assay measuring migration of 231-LUC and BT-549 transfected with miR-NC or miR-630 mimics together with either pcDNA3.1-MTDH or control vector. C. Matrigel invasion assay measuring invasion of 231-LUC and BT-549 transfected with miR-NC or miR-630 mimics together with either pcDNA3.1-MTDH or control vector. D. Colony formation assay in 231-LUC cells and BT-549 cells transfected with miR-NC or miR-630 mimics together with either pcDNA3.1-MTDH or control vector. After transfection of pcDNA3.1-MTDH or control vector for 24 h, these cells were further transfected with miR-NC or miR-630 mimics. Data represent mean +- SD. * P < 0.05; ** P < 0.01; *** P < 0.001. All experiments were repeated independent three times.
Figure 3 MiR-630 targets MTDH directly in breast cancer cell lines A. A schematic diagram illustrating the miR-630-binding sites and 3 mutated bases in MTDH 3'UTR. B. Dual-luciferase assays showing repression of wild-type UTR or mutant UTR following transfection of miR-630 or NC http://mimics.in BT-549 (Left) and HEK293T (Right) cells. Data represent mean +- SD. * P < 0.05; ** P < 0.01; *** P < 0.001; All experiments were repeated independently three times. C. Western blot analysis showing the depression of MTDH in breast cancer cell lines 231-LUC (Left) and BT-549 (Right) transfected with miR-NC or miR-630 with beta-actin as a loading control. D. Western blot describing the MTDH expression in clinical specimens with vinculin as a loading control. The folds change indicated the MTDH expression in tumors against paratumor normalized to vinculin. E. Expression and correlation of miR-630(Log 2 ) and MTDH (log 10 ) in paired clinical breast cancer samples.
Immunohistochemistry (Paraffin) (IHC (P))
Western Blot (WB)
Host / Isotype
Raised against the C-term of human Metadherin.
Antigen affinity chromatography
PBS, pH 7.4
0.1% sodium azide
Metadherin (Metastasis adhesion protein), also known as MTDH, LYsine-RIch CEACAM1 co-isolated (LYRIC), is a novel protein that localizes with the tight junction proteins ZO-1 and occludin in polarized epithelial cells. At the tight junction, it acts not as a structural component, but is rather recruited during the maturation of the tight junction complex. Metadherin is overexpressed in breast cancer tissue and breast tumor xenografts, while much lower levels are expressed in normal breast tissue. Metadherin binds to lung vasculature, one of the four common sites of breast cancer metastasis, through a C-terminal segment in the extracellular domain; blocking this lung-homing domain with antibodies or inhibiting metadherin with siRNA has been reported to inhibit breast cancer metastasis.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Have you cited this antibody in a publication?
so we can reference it in this datasheet.
Protein Aliases: 3D3; 3D3/LYRIC; AEG-1; AEG1; astrocyte elevated gene 1; Astrocyte elevated gene-1 protein; LYRIC; LYRIC/3D3; Lysine-rich CEACAM1 co-isolated protein; Metadherin; Metastasis adhesion protein; MTDH; Protein LYRIC
Gene Aliases: 2610103J23Rik; 3D3; 3D3/Lyric; AEG-1; AEG1; AV353288; D8Bwg1112e; LYRIC; LYRIC/3D3; MTDH
UniProt ID: (Human) Q86UE4, (Mouse) Q80WJ7
Entrez Gene ID: (Human) 92140, (Mouse) 67154
Suggested Secondary Antibodies
Material safety data sheets (MSDS)