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Dach1

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DACH1 is a rabbit polyclonal antibody that recognizes the DACH1 protein. DACH1 is a transcriptional regulator that plays a role in embryonic development and cell differentiation.

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Lab products found in correlation

Cyclin d1, by Santa Cruz Biotechnology (4 mentions) γh2ax, by Merck Group (4 mentions) Ve cadherin, by BD (3 mentions) Vegfr2, by R&D Systems (3 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (3 mentions) β actin, by Proteintech (3 mentions) Vegfr3, by R&D Systems (2 mentions) Sox17, by R&D Systems (2 mentions) Myc tag for coup tf2oe, by Cell Signaling Technology (2 mentions) Cx40a, by Abcam (2 mentions) Cldn11, by Abcam (2 mentions) Anti actin α smooth muscle fitc, by Merck Group (2 mentions) Cxcr4, by BD (2 mentions) Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (2 mentions) Vinculin, by Merck Group (2 mentions) Lamin b1, by Abcam (2 mentions) β actin, by Santa Cruz Biotechnology (2 mentions) β tubulin, by Santa Cruz Biotechnology (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions)

Spelling variants of this product

Anti-DACH1 antibody (2 citations) Anti-DACH1 (2 citations)

15 protocols using dach1

1

Whole-mount Immunostaining Protocol

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Whole-mount immunostaining was performed as previously described18 (link), 52 (link). Antibodies used were: VE-cadherin (BD Biosciences, 550548, 1:250), VEGFR2 (R&D Systems, AF644; 1:250), DACH1 (Proteintech, 10914-1-AP, 1:1000), Myosin (Smooth Muscle) Heavy Chain (Alfa Aesar, BT 562, 1:1000), CTNT (DSHB, CT3-3, 1:1000) and NKX2.5 (Santa Cruz, sc-8697, 1:250). Secondary antibodies were Alexa Fluor conjugates (488, 555, 647, Life Technologies; 1:250).
Rhee S., Chung J.I., King D.A., D’amato G., Paik D.T., Duan A., Chang A., Nagelberg D., Sharma B., Jeong Y., Diehn M., Wu J.C., Morrison A.J, & Red-Horse K. (2018). Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease. Nature Communications, 9, 368.
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2

Western Blot Analysis of Key Signaling Proteins

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Protein isolation and western blot were performed as described previously. [12] Primary antibodies were as following: DACH1 (Proteintech); c-Myc, p21, phospho-Smad3, cyclinD1 (Cell signaling Technology); CDK4 (Affinity); phospho-Smad2 (Millipore); cyclinE1, CDK2, Smad2 and Smad3 (Bioworld Technology); β-Actin (Beyotime). The blots were visualized using enhanced chemiluminescence (Pierce Bioscience).
Wu L., Herman J.G., Brock M.V., Wu K., Mao G., Yan W., Nie Y., Liang H., Zhan Q., Li W, & Guo M. (2014). Silencing DACH1 Promotes Esophageal Cancer Growth by Inhibiting TGF-β Signaling. PLoS ONE, 9(4), e95509.
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3

Immunohistochemical Analysis of Kidney Cancer

Cited 2 times
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Human kidney cancer tissue arrays were purchased from Alenabio (Xi’an, China), including 13 cases of normal renal tissues, 5 cases of cancer adjacent normal renal tissue, 55 cases of clear cell carcinoma tissue, 31 cases of granular cell carcinoma tissue, and 19 cases of transitional cell carcinoma tissue. Tumor tissues were sub-grouped as grade I, well-differentiated; grade II, moderately differentiated; and grade III, poorly or undifferentiated. TNM status was as follows: 66 cases of T1N0M0, 33 cases of T2N0M0, 9 cases of T3N0M0 and 2 cases of T4N0M0. Tissue immunohistochemical stains were performed by core facility using a streptavidin-biotin technique and semi-quantated as described [51 (link)]. The antibodies were polyclonal antibody to DACH1(Proteintech, 10914–1), to PCNA (Santa Cruz, sc-7907) and monoclonal antibody to Cyclin D1 (Santa Cruz, sc-20044). The immune stain intensity and ratio of positive cells were analyzed. Immunofluorescence staining for DACH1 was modified from published method [52 (link)]. Cells were fixed in 4% formaldehyde for 10 minutes, permeable by 1% Triton X-100 for 5 minutes then blocked in 5% goat serum for 1 hour. Primary anti-Flag antibody (M2, Sigma, F3165) was used at 1:200 dilution, the goat anti-mouse secondary antibody (Alexa Fluor-568) was used at 1:500. Cell nuclei were count-stained with 4,6-diamidino-2-phenylindole(DAPI).
Chu Q., Han N., Yuan X., Nie X., Wu H., Chen Y., Guo M., Yu S, & Wu K. (2014). DACH1 inhibits cyclin D1 expression, cellular proliferation and tumor growth of renal cancer cells. Journal of Hematology & Oncology, 7, 73.
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4

Western Blot Analysis of Protein Expression

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Cells were lysed in in RIPA Lysis Buffer (Beyotime Biotechnology, China), and the protein concentration was determined by a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA). The proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for approximately 1.5 h at 90 V-120 V, and then transferred to PVDF membranes (Millipore, USA) at 250 mA for 60 min. After blocked with 5% non-fat milk, the membranes were incubated overnight at 4 °C with specific primary antibodies (FOXM1, 1:200, Santa Cruz Biotechnology, USA; PCDH10, 1:500, Proteintech, China; DACH1, 1:500, Proteintech, China; SMAD4, 1:500, Proteintech, China; β-actin, 1:2000, Proteintech, China). After being washed with TBST (Tris-buffered saline plus Tween-20) three times, the membranes were incubated with corresponding secondary antibodies for 1 h. Bound antibodies were visualized with Li-Cor Odyssey imaging system (Li-COR Biosciences, USA) according to the manufacturer's instructions.
Wang X., Dou N., Wang J., Zhang Y., Li Y, & Gao Y. (2021). FOXM1-induced miR-552 expression contributes to pancreatic cancer progression by targeting multiple tumor suppressor genes. International Journal of Biological Sciences, 17(4), 915-925.
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5

Immunofluorescence Staining of Endothelial Markers

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The following primary antibodies were used at the indicated concentrations: MYC-Tag for COUP-TF2OE (Cell Signaling Technology, Inc., 2278S, 1:300), VE-Cadherin (BD Pharmingen, 550548, 1:125), VEGFR2 (R&D Systems, AF644, 1:125), Cx40 (Alpha Diagnostic International, CX40A, 1:300), ERG (Abcam, ab92513, 1:500), CXCR4 (BD Pharmingen, 551852, 1:125), GFP (Abcam, ab13970, 1:500), VWF (Abcam, ab6994, 1:500), CLDN11 (Abcam, ab53041, 1:1000), SOX17 (R&D Systems, AF1924, 1:500), anti-actin α-smooth muscle- FITC (Sigma, F3777, 1:200), VEGFR3 (R&D Systems, AF743, 1:125), DACH1 (Proteintech, 10914-1-AP, 1:500), JAG1 (R&D Systems, AF599, 1:125).
All secondary antibodies were Alexa Fluor conjugates (488, 555, 633, 635, 594, 647, Life Technologies, 1:125 or 1:250). DAPI (1mg/ml) was used at 1:500.
Su T., Stanley G., Sinha R., D’Amato G., Das S., Rhee S., Chang A.H., Poduri A., Raftrey B., Dinh T.T., Roper W.A., Li G., Quinn K.E., Caron K.M., Wu S., Miquerol L., Butcher E.C., Weissman I., Quake S, & Red-Horse K. (2018). Single cell analysis of early progenitor cells that build coronary arteries. Nature, 559(7714), 356-362.
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6

Western Blotting of Cellular Fractions

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Whole-cell lysates, nuclear lysates, or cytoplasmic lysates were separated by 8%−11% SDS-PAGE gel and the proteins were transferred to a nitrocellulose membrane for Western blotting, as previously described [30 (link), 67 (link)]. The bands were detected using the enhanced chemiluminescence detection system (Thermo Fisher Scientific #34578). The following antibodies were used: DACH1 (#10914–1-AP, Proteintech), γH2AX (#05–636, Millipore, or #80312, Cell Signaling Technology), Vimentin (#5741, Cell Signaling), cyclin D1 (sc-20044, Santa Cruz Biotechnology), p53 (#sc-6243, Santa Cruz Biotechnology), Vinculin (#V9131, Sigma), β-Actin (#sc-47778, Santa Cruz Biotechnology), β-Tubulin (#sc-9104, Santa Cruz Biotechnology), Lamin B1 (#ab16048, Abcam), and GAPDH (#sc-25778, Santa Cruz Biotechnology).
Li Z., Jiao X., Robertson A.G., Sante G.D., Ashton A.W., DiRocco A., Wang M., Zhao J., Addya S., Wang C., McCue P.A., South A.P., Cordon-Cardo C., Liu R., Patel K., Hamid R., Parmar J., DuHadaway J.B., Jones S.J., Casimiro M.C., Schultz N., Kossenkov A., Phoon L.Y., Chen H., Lan L., Sun Y., Iczkowski K.A., Rui H, & Pestell R.G. (2023). The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses. Research Square.
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7

Western Blot Analysis of Cellular Proteins

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Whole-cell lysates, nuclear lysates, or cytoplasmic lysates were separated by 8–11% SDS-PAGE gel and the proteins were transferred to a nitrocellulose membrane for Western blotting, as previously described [33 (link), 77 (link)]. The bands were detected using the enhanced chemiluminescence detection system (Thermo Fisher Scientific #34578). The following antibodies were used: DACH1 (#10914-1-AP, Proteintech), γH2AX (#05-636, Millipore, or #80312, Cell Signaling Technology), Vimentin (#5741, Cell Signaling), cyclin D1 (sc-20044, Santa Cruz Biotechnology), p53 (#sc-6243, Santa Cruz Biotechnology), Vinculin (#V9131, Sigma), β-Actin (#sc-47778, Santa Cruz Biotechnology), β-Tubulin (#sc-9104, Santa Cruz Biotechnology), Lamin B1 (#ab16048, Abcam), and GAPDH (#sc-25778, Santa Cruz Biotechnology).
Li Z., Jiao X., Robertson A.G., Di Sante G., Ashton A.W., DiRocco A., Wang M., Zhao J., Addya S., Wang C., McCue P.A., South A.P., Cordon-Cardo C., Liu R., Patel K., Hamid R., Parmar J., DuHadaway J.B., Jones S.J., Casimiro M.C., Schultz N., Kossenkov A., Phoon L.Y., Chen H., Lan L., Sun Y., Iczkowski K.A., Rui H, & Pestell R.G. (2023). The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses. Oncogene, 42(22), 1857-1873.
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8

Immunofluorescence Double Staining Protocol

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For immunofluorescence double staining, paraffin-embedded specimens were stained using a modified indirect immunofluorescence method. Primary antibodies were selected against DACH1 (Proteintech, Wuhan), Cyclin D1, Cyclin A, p21, p53, PCNA, Bcl-2 and Bax (Santa Cruz Biotechnology, Dallas, TX). DyLight 488and Cy3-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA) were then used. Colocalization was analyzed by immunofluorescence microscopy.
Liu Q.Q., Zhou Y.Q., Liu H.Q., Qiu W.H., Liu H., Hu T.Y., Xu Q., Lv Y.M, & Wu K.M. (2016). Decreased DACH1 expression in glomerulopathy is associated with disease progression and severity. Oncotarget, 7(52), 86547-86560.
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9

Prostate Histopathological Assessment in Mice

Cited 1 time
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Transgenic mice aged 15 weeks were euthanized by CO2 asphyxiation. Animals were dissected, and the following organs were removed: ventral, lateral and anterior prostates (AP) for hematoxylin & eosin (H&E) and ventro-dorsolateral (VDL) prostates for immunohistochemical (IHC) staining. Histopathological grading was undertaken in a blinded manner, comparing a total of 10 mice (5 of each genotype), analyzing sections from the anterior, ventral and lateral prostate in each animal (total N = 15 in each group). DACH1 (#10914-1-AP, Proteintech), Ki-67 (#M7240, Dako), Beclin 1 (#11427, Santa Cruz Biotechnology), AR (N20) (#sc-816, Santa Cruz Biotechnology), SMAD2pSer465/467 (#44-244G, ThermoFisher Scientific), γH2AX (Ser139, 05-636, Millipore), Cleaved Caspase-3 (#9661, Cell Signaling Technology), 53BP1 (NB100-304, Novus Biologicals) antibodies were used as described [76 (link)]. ImageJ software was used in IHC quantification.
Li Z., Jiao X., Robertson A.G., Di Sante G., Ashton A.W., DiRocco A., Wang M., Zhao J., Addya S., Wang C., McCue P.A., South A.P., Cordon-Cardo C., Liu R., Patel K., Hamid R., Parmar J., DuHadaway J.B., Jones S.J., Casimiro M.C., Schultz N., Kossenkov A., Phoon L.Y., Chen H., Lan L., Sun Y., Iczkowski K.A., Rui H, & Pestell R.G. (2023). The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses. Oncogene, 42(22), 1857-1873.
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10

Western Blot and Immunofluorescence Antibodies

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The following antibodies were used for Western blotting and immunofluorescence: YB-1 (Abcam: Ab12148, Proteintech Group: 20339-1-AP, Bethyl Laboratories, Inc (A): A404-230-T, Bethyl Laboratories, Inc (B): A303-231-T), Dach1 (Proteintech Group: 10914-1-AP), Caprin1 (Proteintech Group: 14112-1-AP), G3BP1 (Santa Cruz Biotechnology, Inc: sc-81940), G3BP2 (Bethyl Laboratories, Inc: A302-040A), TIA-1 (Santa Cruz Biotechnology, Inc: sc-1751), TIAR (Santa Cruz Biotechnology, Inc: sc-1749), eIF3b (Santa Cruz Biotechnology, Inc: sc-16377), eIF4G (Santa Cruz Biotechnology, Inc: sc-11373), eIF4E-BP1 (Cell Signaling Technology: 9452S), PABP (Santa Cruz Biotechnology, Inc: sc-32318), Nucleolin (Santa Cruz Biotechnology, Inc: sc-9893), eIF4E (Santa Cruz Biotechnology, Inc: sc-9976), HuR (Santa Cruz Biotechnology, Inc: sc-5261), Fxr2 (Santa Cruz Biotechnology, Inc: sc32266), Rack1 (Santa Cruz Biotechnology, Inc: sc-17754), RPS23 (Santa Cruz Biotechnology, Inc: sc-100837), Twist1 (Bethyl Laboratories, Inc: A301-394A), Snail1 (Origene: TA500416), Zeb1 (Bethyl Laboratories, Inc: A301-921A), Puromycin (EMD Milipore: 12D10), GFP (Applied Biological Materials: G160), β-actin (Proteintech Group: 66009-1).
Lyons S.M., Achorn C., Kedersha N.L., Anderson P.J, & Ivanov P. (2016). YB-1 regulates tiRNA-induced Stress Granule formation but not translational repression. Nucleic Acids Research, 44(14), 6949-6960.
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