Cells were lysed in radioimmunoprecipitation assay buffer (1% Igepal CA 630, 0.5% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Fifty micrograms of total protein was separated on sodium dodecyl sulfate–polyacrylamide gels and transferred onto nitrocellulose membranes (Merck Millipore, Billerica, MA). The blots were probed with antibodies against YAP, phospho-YAP (pYAP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) as well as TEAD1 (Santa Cruz Biotechnology, Dallas, TX). Secondary, HRP-coupled antibodies directed against rabbit or mouse IgG, respectively, were purchased from Cell Signaling Technology. Detection was performed as previously described [13] (link).
Tead1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
TEAD1 is a protein-coding gene that produces the transcriptional enhancer factor (TEF) family of transcription factors. These factors play a role in the regulation of gene expression, but a detailed description of the core function cannot be provided while maintaining an unbiased and purely factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Secondary fluorescent antibodies, by Abcam (2 mentions)
Rock inhibitor y2763, by Merck Group (2 mentions)
Pcdna3, by Addgene (2 mentions)
Shyap1 2, by Addgene (2 mentions)
Gna13, by Santa Cruz Biotechnology (2 mentions)
Rgs12 antibody ab1, by Merck Group (2 mentions)
Pdz domain peptides of rgs12, by GenScript (2 mentions)
Gpcr activator lpa, by Merck Group (2 mentions)
Gpcr inhibitor ki6425, by Merck Group (2 mentions)
Rho gtpases inhibitor c3, by Merck Group (2 mentions)
Tead1 sirna and controls, by Santa Cruz Biotechnology (2 mentions)
Pcdna3.1 gfp yap, by Addgene (2 mentions)
Pet gst yap, by Addgene (2 mentions)
Pcdna3.1 rhoa, by Addgene (2 mentions)
Shrgs12 1, by Applied Biological Materials (2 mentions)
Shrgs12 2, by Applied Biological Materials (2 mentions)
Shrgs12 3, by Applied Biological Materials (2 mentions)
Edta free cocktail inhibitor tablets, by Thermo Fisher Scientific (2 mentions)
Fugene hd transfection reagent, by Promega (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
5 protocols using tead1
1
Western Blot Analysis of YAP and TEAD1
2
Western Blot Analysis of Cellular Proteins
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Total protein was isolated from cells using Cell Extraction Buffer (Biosource, Camarillo, CA) supplemented with protease and phosphatase inhibitors and precleared using centrifugation, followed by measuring protein concentrations using the BCA Protein Assay kit (Pierce, Rockford, IL). The primary antibodies including TAZ, CTGF, Notch1, Hes1, LDHA, PFKB3, PEPCK, PKM2, PGK1, HK2, GLUT1, GLUT3, ALDOA, p-TAZ (Ser89), and β-actin were purchased from Cell Signaling Technology (CA, US). Jagged1 and TEAD1 were purchased from Santa Cruz Biotechnology (CA, US). Appropriate secondary antibodies conjugated to horseradish peroxidase were used, including anti-mouse or anti-rabbit IgG (GE-Healthcare, CA, US). Proteins were visualized by Amersham enhanced chemiluminescence (GE-Healthcare, CA, US). Immunohistochemistry assay was performed as previously described [7 (link)].
3
Immunoblotting and Immunoprecipitation Protocols
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As previous reports [46 (link), 47 (link)], CM cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and quantified by the Bradford method and Ponceau S staining of nitrocellulose membranes. 30 µg protein samples were subjected to 10% SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). Immunoblot membranes were blocked in 5% milk for 1 h. Membranes were incubated with the primary antibody, including CDC42 (Santa Cruz, sc84011, 1:200), TEAD1 (Santa Cruz, sc393976, 1:200), AGO2 (Santa Cruz, sc376696, 1:500), IGF2BP2 (Proteintech, 11601-1-AP, 1:500) and GAPDH (Santa Cruz, sc-47724, 1:1000) at cold room for overnight. After three washes with TBST, membranes were incubated with secondary antibody for 1 h, washed, and exposed to Pierce ECL Western Blotting Substrate (Thermo, #32106) for detection of protein expression.
Proteins were lysed in RIPA buffer and immunoprecipitated with AGO2 or IGF2BP2 antibody and the corresponding IgG. After applying a magnet, proteins associated with Protein A/G Magnetic Beads were washed three times and analyzed by western blotting. The signal densities of protein bands were quantified and normalized to that of GAPDH using the ImageJ software.
Proteins were lysed in RIPA buffer and immunoprecipitated with AGO2 or IGF2BP2 antibody and the corresponding IgG. After applying a magnet, proteins associated with Protein A/G Magnetic Beads were washed three times and analyzed by western blotting. The signal densities of protein bands were quantified and normalized to that of GAPDH using the ImageJ software.
4
Dissecting YAP/TEAD Signaling Pathway
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Antibodies against p127YAP (D9W2I), YAP (D8H1X), MST1, pMST (E7UD1), LATS1 (C66B5), pLATS1, Lamin B1 (D9V6H) and GAPDH were from CST. Antibodies of Ezrin, GNA12, GNA13, RhoA, flag, TEAD1, GST and GFP were from Santa Cruz Biotechnology. RGS12 antibody (ab1) was from Sigma. The secondary fluorescent antibodies were from Abcam. PDZ domain peptides of RGS12 were purchased from GenScript. GPCR activator LPA, GPCR inhibitor Ki6425, Rho GTPases inhibitor C3 and Rock inhibitor Y2763 were obtained from Sigma. Doxorubicin hydrochloride (DOX), methotrexate 4-Amino-10-methylfolic acid hydrate (MTX) and EDTA-free cocktail inhibitor tablets were all obtained from Fisher Scientific™. TEAD1 siRNA and controls were from Santa Cruz Biotechnology. FuGENE® HD Transfection Reagent was purchased from Promega Corporation. The primers used for the quantification are listed in Supplementary Table 1 .
The following plasmids: pRL-TK was generously provided by Dr. Zhen Zhang (University of Pennsylvania, Philadelphia, PA, USA); pcDNA3.1, shYAP1/2, pcDNA3.1-GFP-YAP, PET-GST-YAP and pcDNA3.1-RhoA were obtained from Addgene. Three human shRGS12 lentivectors (shRGS12–1, shRGS12–2 and shRGS12–3; Catalog # i019000) were ordered from ABM. pcDNA3.1-flag-RGS12 (flag-RGS12) and the pcDNA3.1-flag-RGS12 mutant with the deletion of the PDZ domain vectors (flag-RGS12ΔPDZ) were constructed in our lab.
The following plasmids: pRL-TK was generously provided by Dr. Zhen Zhang (University of Pennsylvania, Philadelphia, PA, USA); pcDNA3.1, shYAP1/2, pcDNA3.1-GFP-YAP, PET-GST-YAP and pcDNA3.1-RhoA were obtained from Addgene. Three human shRGS12 lentivectors (shRGS12–1, shRGS12–2 and shRGS12–3; Catalog # i019000) were ordered from ABM. pcDNA3.1-flag-RGS12 (flag-RGS12) and the pcDNA3.1-flag-RGS12 mutant with the deletion of the PDZ domain vectors (flag-RGS12ΔPDZ) were constructed in our lab.
5
Dissecting YAP/TEAD Signaling Pathway
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