mouse anti-human garp  (Enzo Biochem)

Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Evaluation of GARP in HUVEC, LEC, and HLF cells cultured under basal conditions. ( a ) Western blot analysis of GARP expression. The blot is a representative blot out of 4 independent experiments. The bar graph shows the quantification of GARP protein levels relative to the GAPDH protein signal (GARP/GAPDH signals) ( n = 4, means ± SD, n.s. determined by one-way ANOVA). ( b ) Flow cytometry analysis of GARP at the surface of primary cells. Jurkat cells overexpressing GARP (Jurkat−hGARP) were used as a positive control. The isotype control is represented in grey, and the positive signal is depicted in red as a percentage of the maximum. The relative MFI of GARP in flow cytometry is represented with a bar graph ( n ≥ 3, means ± SD, n.s., no significance, determined by one-way ANOVA).

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Cell Culture, Western Blot, Expressing, Flow Cytometry, Positive Control

Journal: Cancers

Article Title: Spatial Distribution of Non-Immune Cells Expressing Glycoprotein A Repetitions Predominant in Human and Murine Metastatic Lymph Nodes

doi: 10.3390/cancers15235621

Figure Lengend Snippet: Full scans of Western blot images shown in a. ( a ) Western blot membrane of GARP in Jurkat hGARP used as a positive control. The red rectangle corresponds to the first lane in a. ( b ) Western blot membrane of GARP on HUVECs, LECs, and HLFs in which the red rectangle corresponds to the right part of Western blot shown in a.

Article Snippet: Sections were blocked with animal-free blocking solution (15019 L, Cell Signaling), followed by an incubation with either 5 µg/mL of mouse monoclonal anti-human GARP antibody (clone MHG-6 [ ]) in Dako antibody diluent or no primary antibody (negative control) for 90 min. After two washes with PBS supplemented with Tween20 (PBS-T), the EnVision-HRP secondary antibody (K4001, Dako Agilent, Diegem, Belgium) was incubated at room temperature for 30 min. Staining was amplified using tyramide signal amplification working solution (TSA, NEL741001KT, PerkinElmer) containing fluorescein isothiocyanate dye (1:500, FITC) for 10 min, washed thrice with PBS-T.

Techniques: Western Blot, Membrane, Positive Control

Journal: Cancer research

Article Title: Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer

doi: 10.1158/0008-5472.CAN-16-1456

Figure Lengend Snippet: (A) IHC validation of the human GARP antibody using murine 4T1 cells expressing human GARP (hGARP). Upper panels: staining of 4T1 cells expressing hGARP with anti-hGARP or control antibody. Lower panels: hGARP antibody was pre-adsorbed with 10 μg of recombinant hGARP (rhGARP) or 10 μg of bovine serum albumin (BSA) for 30 minutes at room temperature prior to the application on the slides. Scale bar: 40 μm. (B) Representative images of GARP IHC (brown color) of breast cancer with their matched normal breast tissues. Each patient specimen in these TMAs was represented in two cores on the slide and each core measured 1 mm in diameter. Scale bar: 50 μm. (C) Expression intensity of GARP-positive cells in breast cancer specimens and normal breast tissues. (D) shRNA knockdown of GARP mRNA in NMuMG* cells. Cells treated with scrambled shRNA (SCR) were used as control. Real-time RT-PCR was performed to quantify mRNA of Lrrc32 using β-actin as a control. MNE: mean normalized expression. (E) Flow cytometric analysis of cell surface GARP expression on GARP KD and SCR NMuMG* cells. (F) Immunoblot of GARP and TGF-β level in GARP KD and SCR NMuMG* whole cell lysates. (G) In vitro cell proliferation assay for GARP KD and SCR NMuMG* cells by MTT assay. (H) NMuMG* SCR and NMuMG*-GARP KD cells were injected into NOD-Rag-1−/− mice, followed by weekly monitoring of the tumor growth kinetics. (I) 120 days after tumor injection, lungs gross metastatic nodules were counted visually on the surface of the organ. **P<0.01. Statistical analysis was determined by 2-way ANOVA or two-tailed T-test, where appropriate. Data are representative of at least two independent experiments.

Article Snippet: The mouse anti-human GARP (hGARP) antibody (ALX-804-867-C100, Enzo Life Sciences) was first verified by Western blot using hGARP-transfected HEK-293 cells and by IHC with hGARP-transfected 70Z/3 cells.

Techniques: Expressing, Staining, Recombinant, shRNA, Quantitative RT-PCR, Western Blot, In Vitro, Proliferation Assay, MTT Assay, Injection, Two Tailed Test

Journal: Cancer research

Article Title: Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer

doi: 10.1158/0008-5472.CAN-16-1456

Figure Lengend Snippet: (A) Surface staining of pre-B cells stably expressing human GARP (pre-B-hGARP) by 4D3 GARP antibody. Grey histogram represents staining with isotype control antibody. (B) pre-B-hGARP cells were incubated without or with human LTGFβ (huLTGF-β), in the presence of GARP antibodies or Isotype control antibody. Cells were then stained for cell surface hLTGF-β, in order to determine the activity of the antibody to block the binding of hLTGF-β to GARP. (C) 4T1-EV (empty vector) cells and 4T1-GARP (human GARP overexpression cells) was stained with 4D3 or a commercial anti-GARP antibody (G14D9), followed by flow cytometry. To determine if G14D9 blocks staining by 4D3 antibody, cells were pre-incubated with 1 or 5 μg/ml G14D9 antibody, followed by 4D3 antibody (1 μg/ml) and appropriate fluorochrome-labeled secondary antibody, and flow cytometric analysis. (D) 4T1-GARP cells were seeded in triplicates in a 96-well plate (2,000 cells per well) with 10 μg/ml 4D3 or mouse IgG followed by MTT assay kinetically. (E) BALB/c mice were injected with 5×105 4T1-hGARP mammary tumors orthotopically in the 4th mammary fat pad, followed by IP injection of 200 μg/mouse of 4D3 GARP antibody every three days. The primary tumor growth kinetics was measured three times a week. Significance is indicated at various time points along the tumor curves. (F) Four weeks after tumor injection, mice were sacrificed. Lungs were isolated and the numbers of visible metastatic nodules were counted. (G) Spleens were harvested and percentage of Treg (FoxP3+CD25+CD4+) cells in the antibody treated mice versus Isotype antibody groups were determined by flow cytometry. (H) BALB/c mice were injected with 5×105 4T1-hGARP mammary tumors orthotopically in the 4th mammary fat pad, followed by one dose of cyclophosphamide (CY) and IP injection of 200 μg/mouse 4D3 GARP antibody every three days. The primary tumor growth kinetics was measured three times a week. Significance is indicated at various time points along the tumor curves. (I) Five weeks after tumor injection, mice were sacrificed, tumors were excised and weighted. (J) Lungs were isolated and numbers of visible metastatic nodules were counted. *P<0.05; **P<0.01; ***P<0.001. Significance was determined by two-tailed Student’s t-test. Results are representative of two independent experiments.

Article Snippet: The mouse anti-human GARP (hGARP) antibody (ALX-804-867-C100, Enzo Life Sciences) was first verified by Western blot using hGARP-transfected HEK-293 cells and by IHC with hGARP-transfected 70Z/3 cells.

Techniques: Staining, Stable Transfection, Expressing, Incubation, Activity Assay, Blocking Assay, Binding Assay, Plasmid Preparation, Over Expression, Flow Cytometry, Labeling, MTT Assay, Injection, Isolation, Two Tailed Test