Cellular extracts were prepared using lysis buffer containing 50 mM Tris HCL (pH 7.5), 250 mM NaCl, 1% NP-40, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, Halt™ Protease, and Phosphatase Inhibitor Cocktail (ThermoFisher Scientific). Extracts were run on a 4–12% Bis-Tris gel (Novex) and transferred onto PVDF membranes. Blots were blocked in 5% milk PBS-T (TBS for phospho-specific) for 1 h at room temperature followed by overnight incubation at 4 ˚C with primary antibodies. HRP-conjugated secondary antibodies were used at 1:10,000. Antibodies used in this study include Nanog (ReproCELL, RCAB001P, 1:1000), Klf2 (Millipore, 09–280, 1:1000), Esrrb (Perseus Proteomics, PP-H6705–00, 1:500), Stat3 (Cell Signaling, 124H6, 1:1000), pStat3 Y705 (Cell Signaling, D3A7, 1:1000), Sox2 (Santa Cruz, sc-17320, 1:250), Tfcp2l1 (R&D, AF5726, 1:250), NHLRC3 (OriGene, TA336106, 1:500), anti-HA (Cell Signaling, C29F4, 1:1000), and Actin-HRP (Santa Cruz, sc-1616, 1:2500). Uncropped scans of the most important blots are included in Supplementary Figure 5 .
Tfcp2l1
Manufactured by R&D Systems
Tfcp2l1 is a transcription factor that binds to the CP2 promoter and regulates gene expression. It is involved in various cellular processes such as development and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Stat3, by Cell Signaling Technology (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Rcab001p, by Merck Group (1 mentions)
Anti ha, by Cell Signaling Technology (1 mentions)
Actin hrp, by Santa Cruz Biotechnology (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Nanog, by ReproCELL (1 mentions)
P stat3 y705, by Cell Signaling Technology (1 mentions)
Halt protease, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor cocktail, by Roche (1 mentions)
Phospho stat3, by Cell Signaling Technology (1 mentions)
Ab19878, by Abcam (1 mentions)
Blimp1, by Santa Cruz Biotechnology (1 mentions)
Tfap2c sc 12762, by Santa Cruz Biotechnology (1 mentions)
Sc 47732, by Santa Cruz Biotechnology (1 mentions)
Af5726, by R&D Systems (1 mentions)
Dmi8 microscope, by Leica (1 mentions)
3 protocols using tfcp2l1
1
Western Blot Characterization of Stem Cell Markers
2
Lysing Embryonic Bodies for Protein Analysis
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EBs were lysed in Pierce IP Lysis Buffer (ThermoScientific, Prod# 87787) supplemented with Roche proteinase inhibitor cocktail (11836153001), sodium fluoride (NaF) and sodium orthovanadate (Na3VO4) to a final concentration of 1 mM. Lysates were prepared per the manufacturer's instructions and used in equivalent amounts for western blotting. Antibodies used: Tfcp2l1 (R&D AF5726), Rac1 (Cytoskeleton ARCO3), Stat3 (Cell Signaling 9139) and phospho-Stat3 (Cell Signaling 9136).
3
Immunofluorescence Staining of Stem Cell Markers
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The cells were fixed in 4% paraformaldehyde for 20 min and then incubated in the blocking buffer (PBS containing 5% BSA and 0.2% Triton X-100) for 2 h. After washing three times with PBS, the cells were incubated overnight at 4 °C under the corresponding antibody. Then, after washing with PBS, cells were incubated with the secondary antibody at 37 °C for 1 h. Finally, the cells were photographed with a Leica DMI8 microscope. The antibodies used are Tfcp2l1(AF5726, 1:100, R&D systems), Stella (ab19878, 1:100, Abcam), Tfap2c (sc-12762, 1:100, Santa Cruz), and Blimp1 (sc-47732, 1:100, Santa Cruz).
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