Cells were harvested and lysed with 1% NP-40 lysis buffer (50mM Tris-HCl, 150mM NaCl, 1% NP-40, 1 mM PMSF, 50 mM NaF, 1mM Na3VO4 and 1X protease inhibitor cocktails (Sigma, P8340)). Proteins were separated in 10% SDS-PAGE gel and transferred onto nitrocellulose membrane. The membrane was blocked with 5% skim milk (BD), incubated with primary antibodies (1:1000) in 5% skim milk at 4°C overnight, washed and incubated with HRP-conjugated secondary antibodies (Beyotime, 1:2000) in 5% skim milk for 1h at RT. The membrane was visualized by enhanced chemiluminescence reagents (Millipore). The following antibodies were used for Western blot: TEAD4 (1:1000; Abcam, ab58310), SIX1 (1:1000; Proteintech, 10,709-1-AP), E-cadherin (BD, 610,181), β-Actin (Sigma, A2228).
Tead4
Manufactured by Abcam
Sourced in United States
TEAD4 is a transcriptional enhancer factor that belongs to the TEA/ATTS domain (TEAD) family of transcription factors. TEAD4 plays a key role in the Hippo signaling pathway, which regulates cell proliferation, differentiation, and apoptosis. TEAD4 acts as a transcriptional activator by binding to the DNA sequence 5'-CATTCC-3' and recruiting co-activators to regulate target gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Skim milk, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Beyotime (1 mentions)
Skim milk, by BD (1 mentions)
E cadherin, by BD (1 mentions)
Myogenin, by Santa Cruz Biotechnology (1 mentions)
Vinculin, by Merck Group (1 mentions)
Myoglobin, by Santa Cruz Biotechnology (1 mentions)
Stat3, by Thermo Fisher Scientific (1 mentions)
Ez magna chip a g chromatin immunoprecipitation kit, by Merck Group (1 mentions)
Q700 sonicator, by Qsonica (1 mentions)
Acetyl histone h4 lys8, by Cell Signaling Technology (1 mentions)
Acetyl histone h4, by Cell Signaling Technology (1 mentions)
Acetyl histone h3 lys18, by Cell Signaling Technology (1 mentions)
Pgc 1a, by Cell Signaling Technology (1 mentions)
Histone h2b, by Cell Signaling Technology (1 mentions)
Acetyl histone h4 lys5, by Cell Signaling Technology (1 mentions)
Histone h2a, by Cell Signaling Technology (1 mentions)
6 protocols using tead4
1
Western Blot Analysis of Protein Markers
2
Western Blot Analysis of Muscle Proteins
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Western blot was performed as described for Co-immunoprecipitation with the following antibody: Maf (Abcam, ab77071, dilution 1/1000), Vinculin (Sigma-Aldrich, V9131, dilution 1/1000), Tead4 (Abcam, ab58310, dilution 1/1000), Klf5 (Proteintech, 21017-1-AP, dilution 1/1000), Myogenin (Santa Cruz Biotechnology, sc-12732, dilution 1/1000), Parvalbumin (Millipore Sigma, MAB1572, clone name: PARV-19, dilution 1/1000), Myoglobin (Santa Cruz Biotechnology, sc-7425, dilution 1/1000).
3
Chromatin Immunoprecipitation of LINC00273 and RBMX Promoters
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EZ‐Magna ChIP™ A/G Chromatin Immunoprecipitation Kit (Millipore) was employed for this experiment. In brief, cells (after LINC00273 overexpression or inhibitor treatment if necessary) were subjected to 4% formaldehyde treatment at 37℃ for 10 min for chromatin protein‐DNA crosslinking. Then crosslinked chromatin was extracted, resuspended in lysis buffer and sonicated with Q700 Sonicator (Qsonica, Newtown, CT, USA) into small fragments (500‐1000 bp). Next, for immunoprecipitation, chromatin fragments were cocultured with antibody against STAT3 (Invitrogen), TEAD1 (LSBio), TEAD4 (Abcam), YAP (Invitrogen), or IgG (Abcam) conjugated on magnetic beads at 4℃ overnight. After washing and DNA purification, the quantification of immunoprecipitated LINC00273 promoter (for macrophages) or RBMX promoter (for A549 or H1975 cells) was conducted by quantitative PCR.
4
Comprehensive Protein Analysis Protocol
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TEAD4 (#ab58310), pan-OXPHOS (#ab110413), and Ki67 (#ab15580) were purchased from Abcam. p-mTOR (T2446) (#09-345) and acetyl-histone H3 (#06-599) were purchased from Millipore. Phosphor-PGC1a (#AF6650) was purchased from R&D. mTOR (#2938), p-mTOR (S2448) (#5536), YAP (#14074), p-YAP (S127) (#13008), p-YAP(S397) (#13619), p38 (#8690), p-p38 (T180/182) (#4511), acetyl-histone H3 (Lys9) (#9649), acetyl-histone H3 (Lys14) (#7627), acetyl-histone H3 (Lys18) (#13998), acetyl-histone H3 (Lys27) (#8173), acetyl-histone H3 (Lys56) (#4243), histone H3 (#4499), acetyl-histone H2A (Lys5) (#2579), acetyl-histone H2B (Lys5) (#12799), histone H2A (#12349), histone H2B (#12364), acetyl-histone H4 (Lys5) (#8647), acetyl-histone H4 (Lys8) (#2594), acetyl-histone H4 (Lys12) (#13944), acetyl-histone H4 (#2935), PGC-1a (#2178), p21 (#2974), and p27 (#83630) were purchased form Cell Signaling. All antibodies were used at a dilution of 1:1000 for immunoblotting and 1:250 for immunofluorescent staining.
5
Western Blot Analysis of Hippo Pathway Proteins
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A total of 0.05 μg tissue samples were collected using RIPA lysis buffer (Millipore, Billerica, MA, USA) containing protease inhibitors (Sigma, Shanghai, China). Protein samples were centrifuged and collected. The BCA method (Pierce, Rockford, IL, USA) was used to determine the concentration according to manufacturer’s protocol (Beyotime, Nanjing, Jiangsu, China). Subsequently, proteins as well as loading buffer were heated, after which proteins were separated by 10% SDS electrophoresis and transferred to polyvinylidene fluoride membranes (GVS Technology Co., Ltd., Suzhou, China). After blocked with milk at room temperature for one hour, membranes were incubated in a 1:1000 solution of primary antibodies (MST, MST2, LATS1, LATS2, YAP1, TAZ, TEAD1, TEAD2, TEDA3, TEAD4, GAPDH; Abcam, Cambridge, MA, USA) at 4 °C overnight. Subsequently, membranes were incubated with the secondary antibody at room temperature for one hour and observed using the ECL method (Pierce, Rockford, IL, USA).
6
ChIP-seq Protocol for Transcription Factors
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ChIP experiments were conducted as previously described (23 (link)). Briefly, TSCs were fixed with 1% formaldehyde for 7 min at room temperature, and then glycine (final 125 mM) was added to quench formaldehyde (5 min). The fixed cells were sonicated using a Bioruptor (Diagenode) with a setting of 30 s on and 1 min off for 10 min (total of three times), and the sheared chromatins that have an average of 250 bp DNA fragments were utilized for immunoprecipitation using 10 μg of a native antibody. The antibodies used include EP300 (Santa Cruz, sc-585), FOS (Santa Cruz, sc-7202X), GATA2 (Santa Cruz, sc-9008X), MAFK (Santa Cruz, sc-477X), TEAD4 (Abcam, ab58310), TFAP2C (Santa Cruz, sc-8977) and H3K27ac (Active Motif, 39133). Enriched ChIP materials were used to generate next-generation sequencing libraries using an NEB ChIP-seq library preparation kit (NEB, E7370L). ChIP-seq libraries were sequenced using an Illumina HiSeq 2500 machine.
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