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Ep823y

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EP823Y is a laboratory equipment product. It is a tool used in research and scientific applications, but its core function is not available without the risk of extrapolation or interpretation.

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Lab products found in correlation

Chemidoc imaging system, by Bio-Rad (4 mentions) Sk 4100, by Vector Laboratories (2 mentions) Clarity western ecl substrate, by Bio-Rad (2 mentions) Nitrocellulose membrane, by Merck Group (2 mentions) Phosstop with complete, by Merck Group (2 mentions) Can get signal immunostain solution b, by Toyobo (2 mentions) Tcs sp5 confocal microscope, by Leica camera (2 mentions) Anti β actin, by Santa Cruz Biotechnology (1 mentions) Anti rabbit igg, by Cell Signaling Technology (1 mentions) Cs 7074, by Cell Signaling Technology (1 mentions) Complete ultra tablets and phosstop, by Roche (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Anti icam 1, by Abcam (1 mentions) Vectra3 slide scanner, by PerkinElmer (1 mentions) Anti vcam 1, by Abcam (1 mentions) Ultraview dab detection kit, by Roche (1 mentions) Epr5047, by Abcam (1 mentions) Epr4776, by Abcam (1 mentions) Discovery ultra autostainer, by Roche (1 mentions) Anti p smad3, by Abcam (1 mentions)

13 protocols using ep823y

1

Quantifying Smad3 and NfκB Activation

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For Western blots, a monoclonal rabbit anti-Smad3 (phospho S423/S425) antibody (EP823Y) (ab52903) was used that detects phosphorylated Smad1 and 3 (Abcam). To analyze the activation of the NfκB pathway, phosphorylation of p65 was detected using a monoclonal rabbit antibody to pp65 (D14E12) from Cell Signaling. For loading control, a monoclonal mouse anti-β-actin was used (Santa Cruz Biotechnology).
Gaitantzi H., Karch J., Germann L., Cai C., Rausch V., Birgin E., Rahbari N., Seitz T., Hellerbrand C., König C., Augustin H.G., Mogler C., de la Torre C., Gretz N., Itzel T., Teufel A., Ebert M.P, & Breitkopf-Heinlein K. (2020). BMP-9 Modulates the Hepatic Responses to LPS. Cells, 9(3), 617.
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2

Quantifying p-Smad-2/3 Staining in MC38 Tumors

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For p-Smad-2/3 stain on fresh frozen MC38 tumors, 5 μm tumor sections were fixed with 4% paraformaldehyde followed by incubation with 3% H2O2. To minimize nonspecific staining, sections were incubated with the appropriate animal serum for 20 min at room temperature, followed by incubation with primary anti-p-Smad-2/3 antibody (EP823Y; Abcam) overnight at 4°C. Staining with secondary antibodies (Vectastain ABC Kit) was then performed before development using DAB substrate (Vector Labs SK-4100). The staining intensity of p-Smad-2/3 was graded as follows with the sample identity blinded (0: negative; 1: faint; 2: moderate; 3: strong but less intense than 4; and 4: intense).
Rachidi S., Metelli A., Riesenberg B., Wu B.X., Nelson M.H., Fugle C.W., Paulos C.M., Rubinstein M.P., Garrett-Mayer E., Hennig M., Bearden D.W., Yang Y., Liu B, & Li Z. (2017). Platelets Subvert T Cell Immunity Against Cancer via GARP-TGFβ Axis. Science immunology, 2(11), eaai7911.
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3

Gingival Fibroblast Signaling Pathway Analysis

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Gingival fibroblasts seeded at 30,000 cells/cm2 were serum-starved overnight. Cells were exposed for 30 min to 30% of lysates prepared from PPP, heated PPP (Alb-gel), buffy coat C-PRF, and 5% lysates of the red clot. Protein extracts in SDS buffer containing protease and phosphatase inhibitors (cOmplete ULTRA Tablets and PhosSTOP; Roche, Mannheim, Germany) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, General Electric Company, Fairfield, CT, USA). The binding of rabbit p-Smad3 antibody (phospho S423 + S425, [EP823Y], Abcam, Cambridge, UK) was detected with an antibody labelled with peroxidase (CS-7074, anti-rabbit IgG, Cell Signaling Technology), respectively. Peroxidase was visualized with Clarity Western ECL Substrate (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and signals detected with the ChemiDoc imaging system (Bio-Rad Laboratories).
Kargarpour Z., Nasirzade J., Panahipour L., Miron R.J, & Gruber R. (2020). Liquid Platelet-Rich Fibrin and Heat-Coagulated Albumin Gel: Bioassays for TGF-β Activity. Materials, 13(16), 3466.
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4

Immunohistochemical Analysis of GARP and pSmad-2/3

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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. Both analyses demonstrated specificity of the antibody and dilutions used from 1:250 (colon cancer) to 1:60 (all other cancers).
TMA slides were processed and antigen retrieved as described previously (33 (link)). For mouse IHC, tissue was either placed into OCT media for fresh frozen sections or fixed in 4% paraformaldehyde overnight. Fixed tissue was incubated in 70% ethanol overnight prior to paraffin embedding, and then cut for hematoxylin and eosin (H&E) staining. For p-Smad-2/3 on fresh frozen tumor sections, 5 μm sections were fixed with 4% paraformaldehyde followed by incubation with 3% H2O2. To minimize nonspecific staining, sections were incubated with the appropriate animal serum for 20 min at room temperature, followed by incubation with primary anti-p-Smad-2/3 antibody (EP823Y; Abcam) overnight at 4°C. Staining with secondary antibodies (Vectastain ABC Kit) was then performed before development using DAB substrate (Vector Labs SK-4100). The staining intensity of GARP and pSmad-2/3 was graded as follows with the sample identity blinded (0: negative; 1: faint; 2: moderate; 3: strong but less intense than 4; and 4: intense).
Metelli A., Wu B.X., Fugle C.W., Rachidi S., Sun S., Zhang Y., Wu J., Tomlinson S., Howe P., Yang Y., Garrett-Mayer E., Liu B, & Li Z. (2016). Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer. Cancer research, 76(24), 7106-7117.
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5

Optimized Immunohistochemistry Staining Protocol

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Staining was performed using a Discovery Ultra Autostainer (Ventana). For each immunohistochemistry (IHC) Ab, positive control tissue was initially used for optimization and identification of staining morphology, typically with archival paraffin-embedded tonsil, thymus, bone marrow, or spleen. Optimal Ag retrieval, primary Ab dilution, and detection kits were then determined by staining 4-µm sections of intestine. All Ag retrieval was optimal using a Standard Cell Conditioning 1 buffer (Ventana) retrieval protocol for 64 min at 100°C. The following anti-human Abs were optimized: anti-pSMAD3 (phosphor-S423 and S425, rabbit monoclonal clone EP823Y, dilution 1:100; Abcam), anti–MAdCAM-1 (mouse monoclonal, clone 355G8; Thermo Fisher Scientific, dilution 1:100), anti–ICAM-1 (rabbit monoclonal, clone EPR4776, dilution 1:400; Abcam), and anti–VCAM-1 (rabbit monoclonal, clone EPR5047, dilution 1:250; Abcam). The Ultraview DAB Detection Kit (Ventana) was used for all Abs. Digital images were acquired using a Vectra 3 Slide Scanner (PerkinElmer) for cell adhesion molecules and an BX43 microscope (Olympus) for pSMAD3 IHC. Unless otherwise noted, representative images were selected from 10 samples stained in each group.
Keir M.E., Fuh F., Ichikawa R., Acres M., Hackney J.A., Hulme G., Carey C.D., Palmer J., Jones C.J., Long A.K., Jiang J., Klabunde S., Mansfield J.C., Looney C.M., Faubion W.A., Filby A., Kirby J.A., McBride J, & Lamb C.A. (2021). Regulation and Role of αE Integrin and Gut Homing Integrins in Migration and Retention of Intestinal Lymphocytes during Inflammatory Bowel Disease. The Journal of Immunology Author Choice, 207(9), 2245-2254.
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6

Evaluating Smad Signaling in Skin Equivalents

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Full-thickness 3D tissue skin equivalents (EpiDermFT, MatTek) were treated with 10 ng/ml and 100 ng/ml rGDF11 or 50 ng/ml TGFβ1 (R&D Systems, Minneapolis, MN) added into proprietary growth factor-free media provided by the manufacturer for 2 hours. The treated tissues were placed on ice, washed with ice-cold PBS, lysed in 600μl T-PER Tissue Protein Extraction Reagent (ThermoFischer Scientific), and reacted with anti-P-Smad3 clone (EP823Y, Abcam, Cambridge, MA) and anti-S6 clone (5G10, Cell Signaling, Danvers, MA). The P-Smad2/3 and ribosomal S6 levels were assessed by western blot analysis. Ribosomal S6 was used as a loading control (Fig 3A). Primary human dermal microvascular endothelial cells (HDMEC, PromoCell, Heidelberg, Germany) were grown in Endothelial Growth Cell Medium MV with supplements (PromoCell, Heidelberg, Germany) and seeded for experiment at density of 100,000 cells/5ml into each well of a 6-well plate (triplicate for each treatment). After overnight incubation, cells were transferred into media without supplement and treated with 10 ng/ml, 50 ng/ml, and 100 ng/ml rGDF11 or 50 ng/ml TGFβ1 for 60 minutes. The treated cells were placed on ice, washed with ice-cold PBS, and lysed in 200 μl M-PER Mammalian Protein Extraction Reagent (ThermoFischer Scientific). The P-Smad2/3 and ribosomal S6 levels were analyzed as above (Fig 3B).
Idkowiak-Baldys J., Santhanam U., Buchanan S.M., Pfaff K.L., Rubin L.L, & Lyga J. (2019). Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology. PLoS ONE, 14(6), e0218035.
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7

Western Blot Analysis of Phosphorylated Smad3

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Gingival fibroblasts were serum-starved for 24 h and then preincubated for 30 min with 1% casein and whey powder. Cell extracts containing SDS buffer and protease inhibitors (PhosSTOP with cOmplete; Sigma, St. Louis, MO, USA) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT, USA). Membranes were blocked and the binding of the first antibody raised against p-smad3 (rabbit; phospho S423 + S425; EP823Y, Abcam, Cambridge, UK) and the smad3 (mouse; Smad3 (38-Q): sc-101154, Santa Cruz Biotechnology, SCBT, Santa Cruz, CA, USA) were detected with the appropriate secondary antibody linked to a peroxidase. Chemiluminescence signals were visualized with the ChemiDoc imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Panahipour L., Husejnovic S., Nasirzade J., Semelmayer S, & Gruber R. (2021). Micellar Casein and Whey Powder Hold a TGF-β Activity and Regulate ID Genes In Vitro. Molecules, 26(2), 507.
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8

Phosphorylated Smad-3 Expression in Gingival Fibroblasts

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Gingival fibroblasts were serum-starved overnight and then preincubated for 30 min with 5% human milk. Cell extracts containing SDS buffer and protease inhibitors (PhosSTOP with cOmplete; Sigma, St. Louis, MO) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Whatman, GE Healthcare, General Electric Company, Fairfield, CT). Membranes were blocked and the binding of the first antibody raised against psmad-3 (rabbit; phospho S423 + S425; EP823Y, Abcam, Cambridge, UK) and the Smad-3 (mouse; Smad-3 (38-Q): sc-101154, Santa Cruz Biotechnology, SCBT, Santa Cruz, CA, USA), was detected with the appropriate secondary antibody linked to a peroxidase. Chemiluminescence signals were visualized with the ChemiDoc imaging system (Bio-Rad Laboratories, Inc., Hercules, CA).
Panahipour L., Moghaddam D.M., Nasirzade J., Kargarpour Z, & Gruber R. (2021). RNAseq of TGF-β receptor type I kinase-dependent genes in oral fibroblast exposed to milk. BMC Oral Health, 21, 581.
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9

Cell Lysis and Western Blot Analysis

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Cells were harvested by trypsin-EDTA when necessary, washed in PBS, and lysed on ice in radio-immunoprecipitation assay (RIPA) lysis buffer in the presence of a protease inhibitor cocktail (Sigma-Aldrich). Nuclear-free protein lysate was quantified by Bradford assay (Bio-Rad), and an equal amount of lysate was analyzed by SDS-PAGE and Western blot under reducing conditions using anti–mouse GARP (AF6229; R&D system), anti-mouse Vimentin (D21H3; Cell signaling), anti-mouse E-Cadherin (24E10; Cell Signaling) and anti-mouse p-Smad-2/3 (EP823Y; Abcam) antibodies.
Metelli A., Wu B.X., Fugle C.W., Rachidi S., Sun S., Zhang Y., Wu J., Tomlinson S., Howe P., Yang Y., Garrett-Mayer E., Liu B, & Li Z. (2016). Surface Expression of TGF-β Docking Receptor GARP Promotes Oncogenesis and Immune Tolerance in Breast Cancer. Cancer research, 76(24), 7106-7117.
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10

Analysis of Gingival Fibroblast Signaling

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Gingival fibroblasts were seeded at a density of 50,000 cells/cm2 in 6-well plates and serum-starved overnight. The following day, the cells were exposed to 30% lysates from PRF and UBC for 30 min. Extracts containing SDS buffer with protease and phosphatase inhibitors (Roche, Mannheim, Germany) were separated by SDS–PAGE and transferred to PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were blocked, and the binding of the rabbit phospho S423 + S425 (p-Smad3), (EP823Y, 1:1000, Abcam, Cambridge, UK), Smad3 (CS-9513, 1:1000, Cell Signaling Technology) and actin (sc-47778, 1:1000, Santa Cruz Biotechnology, SCBT, Santa Cruz, CA, USA) primary antibodies was detected with the HRP-labeled secondary antibody (CS-7074, anti-rabbit IgG, and CS-7076, anti-mouse IgG, both 1:10,000, Cell Signaling Technology). After exposure to the Clarity Western ECL Substrate (Bio–Rad Laboratories, Inc., Hercules, CA, USA), chemiluminescence signals were visualized with a ChemiDoc imaging system (Bio–Rad Laboratories). For densitometric analysis, images were analyzed using Image Lab software (Bio–Rad Laboratories).
Kargarpour Z., Panahipour L., Miron R.J, & Gruber R. (2022). Blood Clots versus PRF: Activating TGF-β Signaling and Inhibiting Inflammation In Vitro. International Journal of Molecular Sciences, 23(11), 5897.
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