The protein expressions of ETS1, MDR1, and apoptotic proteins were measured by western blot. Total cell lysates were generated by RIPA lysis buffer (Beyotime, China) and quantified with a BCA kit (Thermo Fisher Scientific, USA). An equal amount of total protein was resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and then transferred to PVDF membrane (Bio-Rad, USA). The membranes were then blocked with 5% non-fat milk at 37°C for 1 h and incubated with the following primary antibodies: Anti-ETS1 (sc55581), anti-MDR1 (sc55510), anti-Bcl-2 (sc7382), anti-Bax (sc7480), and anti-GAPDH (sc47724; Santa Cruz Biotechnology, USA). Finally, the membranes were incubated with secondary antibodies and visualized by an electrochemiluminescence system (Amersham, USA).
Anti ets1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Ets1 is a primary antibody product offered by Santa Cruz Biotechnology. It is designed to detect the Ets1 protein, which is a transcription factor involved in various cellular processes. The antibody can be used in applications such as Western blotting, immunohistochemistry, and immunofluorescence to study the expression and localization of the Ets1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Anti e2f1, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by GeneTex (2 mentions)
Electrochemiluminescence system, by Cytiva (1 mentions)
Anti mdr1, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti bax, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Anti hif 1α clone 54, by BD (1 mentions)
Anti hif 1β, by Santa Cruz Biotechnology (1 mentions)
Anti b23, by Santa Cruz Biotechnology (1 mentions)
Anti vinculin, by Cell Signaling Technology (1 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (1 mentions)
Anti nrf1, by Santa Cruz Biotechnology (1 mentions)
Pcr clean up kit, by Promega (1 mentions)
Anti oct4, by GeneTex (1 mentions)
Anti c myc, by Santa Cruz Biotechnology (1 mentions)
Anti twist, by GeneTex (1 mentions)
8 protocols using anti ets1
1
Protein Expression Analysis by Western Blot
2
Axud1 Interactions with Neural Plate Borders
3
Western Blot Analysis of HGF and Hypoxia Signaling
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(i) The 1833 and MDA-MB231 cells were starved and treated with 100 ng/mL of HGF (R&D System) [14 (link)], or were cultured in 10% FBS and exposed to hypoxia. At various times thereafter, nuclear and total protein extracts were prepared and used for Western blot analysis [30 (link)]. (ii) Some cells cultured with 10% FBS were exposed to hypoxia starting 6 h after miR-125b transfection, and were collected at various times thereafter; some starved cells were exposed to HGF for 24 h at the end of the 48-h treatment with miR-125b. Hypoxia (1% oxygen) exposure was performed as reported before [30 (link)]. All these cells were used for total protein extracts and Western blot assay. The hybridization of the Western blots was performed with the following antibodies: anti-Ets1 (1:2000, C20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-HIF-1α (clone 54) (1:350, BD-Transduction Laboratories, Franklin Lakes, NJ, USA), anti-HIF-1β (1:200, N19, Santa Cruz Biotechnology), anti-Endothelin-1 (50 ng/mL, Calbiochem® Merck Chemicals Ltd., Nottingham, UK), anti-B23 (1:1000, H106, Santa Cruz Biotechnology), anti-vinculin (1:1000, 4650, Cell Signaling Technology, Beverly, MA, USA). Densitometric analysis was performed after reaction with Enhanced chemiluminescence (ECL) kit from Thermo Fisher Scientific.
4
ChIP Assay for Transcription Factor Binding
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After the transfection of the empty vector or the ETS-1, E2F-1, NRF-1, or C/EBPβ plasmid (5 μg) into HEK293 cells, the cells were fixed with 1% (w/v) formaldehyde for 10 min at 25 °C. ChIP was performed, as described previously [7 (link)]. The cells were incubated in 200 µL of sonication buffer and sonicated for 70 s (10 s pulse and 60 s rest). DNA was sheared to lengths between 200 and 1000 bp. The sonication mixture was incubated with mock immunoglobulin G (Santa Cruz Biotechnology, CA, USA), anti-E2F-1 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-NRF-1 (Santa Cruz Biotechnology, CA, USA), anti-ETS-1 (Santa Cruz Biotechnology, CA, USA), or anti-C/EBP antibody (Santa Cruz Biotechnology, CA, USA), followed by immunoprecipitation using Protein A and G agarose bead mixture (Invitrogen, Paisley, UK). The protein–DNA complexes were separated from the beads by elution buffer (50 mM Tris HCl, pH 8.0, 1 mM EDTA, 1% [w/v] sodium dodecyl sulfate (SDS), 50 mM NaHCO3), and then heated at 65 °C for 5 h with protease K to reverse the formaldehyde crosslinks. Finally, the DNA was purified with the PCR Clean-up Kit (Promega, WI, USA). ChIPed DNA was used as a template for PCR (Forward: 5′-GCCTACTACACCAGCC-3′, Reverse: 5′-CATGTTTGGAATGGCG-3′).
5
Whole-Cell Extract Analysis of Stemness Markers
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As described previously [44 (link)], whole-cell extracts were prepared for Western blotting using anti-Notch2 C-terminal, anti-E2F1, anti-c-Myc, anti-E-cadherin, anti-Ets1, anti-plakoglobin (Santa Cruz), anti-vimentin (Sigma), anti-N-cadherin (BD Biosciences), anti-CD44, anti-Nanog, anti-Oct4, anti-SOX2, anti-Twist, and anti-GAPDH (GeneTex) antibodies.
6
ChIP Assay of Transcription Factors
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Chromatin immunoprecipitation (ChIP) was performed according to the EpiQuik™ Chromatin Immunoprecipitation Kit (Epigentek, NY, USA), as previously described [29 (link)]. Briefly, cells were cross-linked with 1% formaldehyde. The cross-linked lysate was sonicated 10 times for 15 s interspersed by 30 s of rest on ice between each pulse to obtain average DNA fragment sizes ranging from 200 to 1000 bp. The sheared DNA was immunoprecipitated with the kit-provided Non-Immune IgG negative control, 4 µg of anti-ETS-1 (Santa Cruz, CA, USA), anti-GRβ (Abcam, CB, GB) and anti-STAT4 (Genetex International, CA, USA). The immunoprecipitated DNA quantification was performed amplifying the region of interest (from − 371 to − 255, human CAT promoter region location from ATG) using qPCR. The primers used were: CAT Chip F, 5′-AGGATGCTGATAACCGGGAG-3′; CAT Chip R, 5′-AGGGTGCGGAAAGGAAGG-3′. The thermal cycle reaction was performed as follows: 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The average cycle threshold of each triplicate was normalized to the input (un-immunoprecipitated DNA). Data are expressed as a percentage of input DNA that represents the enrichment of TFs on the specific region of CAT promoter surrounding rs1001179 SNP.
7
Western Blotting of Cellular Proteins
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Whole-cell extracts were prepared and then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as described27 (link). Western blot analyses were performed with anti-Notch1 C terminal, anti-Ets-1, anti-DNMT3B (Santa Cruz), anti-cleaved Notch1, anti-ERK1/2, anti-phospho-ERK1/2 (Cell Signaling Technology), anti-TRPA1 (Novus Biologicals), anti-vimentin (Thermo Fisher Scientific), and anti-GAPDH (Genetex) antibodies.
8
Western Blotting of Key Signaling Proteins
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Western blotting was carried out as described previously [37 (link)]. The following primary antibodies were used for western blotting: anti-MED23 (BD, 550492), anti-MED16 (Bethyl, A303-668A, RRID: AB_11205632), anti-MED24 (Bethyl, A301-472A, RRID: AB_999675), anti-phosphorylated-Erk1/2 (Thr202/Tyr204) (Santa Cruz, sc-16982), anti-Erk (K-23) (Santa Cruz Biotechnology, sc-94-G, RRID: AB_631456), anti-phosphorylated-Akt (S473) (Cell signaling, 9271), anti-Akt (Cell signaling Technology, 9272, RRID: AB_329827), anti-Ets1 (Santa Cruz, sc-350x), anti-Sprouty2 (Santa Cruz, sc-30049), anti-Src (Cell signaling Technology, 2108s, RRID: AB_331137) and β-Actin (Sigma, A5441).
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