The antibodies used for immunoblotting are listed below: Tead1 [EPR3967(2)] (ab133533, Abcam; 1:2,000), Hsp90 (4874S, Cell Signaling; 1:2,000), Cell Cycle Regulation Antibody Sampler Kit (9932, Cell Signaling; 1:1,000 for all) & Sampler Kit II (9870, Cell Signaling; 1:1,000 for all). The antibodies for immunofluorescence are: cardiac Troponin T [1C11] (ab8295, Abcam; 1:500), Tead1 [EPR3967(2)] (ab133533, Abcam; 1:100), phospho-Histone H3 (Ser10) (9701, Cell Signaling; 1:100), Ki67 (ab16667, Abcam, 1:200).
Ab133533
Manufactured by Abcam
Sourced in United States
Ab133533 is a lab equipment product offered by Abcam. It is a device designed for use in scientific research applications. The core function of this product is to facilitate specific laboratory processes, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab110641, by Abcam (1 mentions)
Ab283575, by Abcam (1 mentions)
Ab92536, by Abcam (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Yeasen (1 mentions)
Ab92547, by Abcam (1 mentions)
Bca kit, by Beyotime (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Ab8295, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Cell cycle regulation antibody sampler kit, by Cell Signaling Technology (1 mentions)
Ab58310, by Abcam (1 mentions)
Chromatin ip kit, by Cell Signaling Technology (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab70561, by Abcam (1 mentions)
Ab62751, by Abcam (1 mentions)
Ab56701, by Abcam (1 mentions)
Ab224239, by Abcam (1 mentions)
Ab13847, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
12 protocols using ab133533
1
Antibody Validation in Cell Cycle Studies
2
Cellular Fractionation and Protein Analysis
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Cellular fractions were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (20126ES50; Yeasen, Shanghai, China). The cells were washed with 1× PBS and lysed in RIPA lysis buffer containing protease inhibitors, and the protein levels in the lysates were quantified using an enhanced bicinchoninic acid assay (BCA) kit (P0012, Beyotime, Shanghai, China). Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and immunoblotted with the indicated antibodies. For immunoprecipitation assays, protein lysates were incubated with anti-ARID1A, anti-YAP, anti-BRG, anti-TEAD, anti-Flag, or normal IgG antibody at 4 °C overnight with rotation. On day 2, the mixture was incubated with protein A/G beads at room temperature for 2–3 h. After washing three times with lysis buffer, the beads were boiled and subjected to Western blotting. The antibodies used were as follows: ARID1A (ab272905, Abcam, Cambridge, UK); YAP (#14074, CST, Fall River, MA, USA); p-YAP (#13008, CST); BRG1 (ab110641, Abcam); TEAD (ab133533, Abcam); E-cadherin (#14472, CST); N-cadherin (#13116, CST); MMP2 (ab92536, Abcam); MMP-9 (ab283575, Abcam); Snail (#3879, CST); Vimentin (ab92547, Abcam); GAPDH (60004-1-Ig, Proteintech, Chicago, IL, USA); Histone H3 (ab17917, Abcam), Flag (20543-1-AP, Proteintech).
3
Chromatin Immunoprecipitation (ChIP) Assay
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Cell extraction was prepared using Chromatin IP kit (Cell Signaling Technology) according to the manufacturer’s protocol. Briefly, 1 × 107 cells were cross-linked with 37% formaldehyde, collected and digested to produce chromatin fragments for incubation with IgG (Cell Signaling Technology) or specific antibodies for Tead1 (ab133533, Abcam, Cambridge, MA, USA), Tead2 (H00008463-M01A, Abnova, Taipei, Taiwan), Tead4 (ab58310, Abcam) and YAP (#4912, Cell Signaling Technology) respectively. ChIP DNA was amplified and analyzed by qPCR and sequencing. Relative enrichment of specific factors was assessed by the formula provided in the protocol. ChIP primers are listed in the Additional file 1 : Table S3.
4
Protein Expression Analysis in Cells
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Antibodies: YAP (ab56701, abcam, 1:5000), Lats1 (ab70561, abcam,1:5000), p-YAP (ab62751, abcam,1:1000), Bax (ab32503, abcam,1:5000), Bcl-2 (ab196495, abcam,1:1000), TAZ (ab224239, abcam,1:5000), Caspase-3 (ab13847, abcam,1:500), GAPDH (ImmunoWay, YM3029,1:5000), Mst1 (CST,3682s,1:1000), TEAD1 (ab133533, abcam,1:5000), Cleaved-caspase3 (CST,9664s,1:1000), Phospho-Lats1/Lats2 (PA5-64591,Invitrogen,1:1000), Histone H3 (ImmunoWay, YM3038,1:5000).
Briefly, protein concentrations of tissues or cells were quantified by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Sample of proteins (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to PVDF membrane, then blocked by 5% non-fat milk in PBST buffer (PBS buffer containing 0.05% Triton-100) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4°C, after 3 times washing in PBST buffer, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Signals were detected by using an ECL substrate (Thermo Scientific) and exposure with the Tanon 5500 imaging system. The intensity of the bands was quantified by densitometry.
Briefly, protein concentrations of tissues or cells were quantified by BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Sample of proteins (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel and transferred to PVDF membrane, then blocked by 5% non-fat milk in PBST buffer (PBS buffer containing 0.05% Triton-100) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at 4°C, after 3 times washing in PBST buffer, the membranes were incubated with the secondary antibodies for 1 h at room temperature. Signals were detected by using an ECL substrate (Thermo Scientific) and exposure with the Tanon 5500 imaging system. The intensity of the bands was quantified by densitometry.
5
Western Blot Analysis of TEAD1 and HIF-1α
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The cell lysate was created by treating the cells with RIPA buffer from Epizyme in Shanghai, China, and the amount of protein was measured using the BCA Protein Quantification Kit. A 30 μg protein sample was subjected to electrophoretic separation on a 12% SDS-polyacrylamide gel. After electrophoresis, the proteins were moved to PVDF membranes. Following incubation with a 5% skim milk solution in TBS-T for four hours at room temperature, primary antibodies against TEAD1 (diluted 1:2000, purchased from Abcam, catalog number ab133533, UK) and HIF-1α (diluted 1:2000, acquired from Abcam, catalog number ab79546, UK) were left overnight at 4°C. To ensure equal loading of protein samples, an anti-β-actin antibody (diluted at 1:5000, sourced from Abcam, catalog number ab6275, United Kingdom) was employed. After washing the membranes three times with TBS-T for 15 minutes each, they were then incubated with secondary antibodies (diluted at 1:10,000; Westang) conjugated to horseradish peroxidase for 45 minutes at room temperature. Protein bands were visualized using a developing solution (Vazyme, Nanjing, China) and exposed to X-ray film.
6
Chromatin Immunoprecipitation and Protein-DNA Interaction Profiling
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DNA and proteins were crosslinked in cells using formaldehyde (Sinopharm Chemical Reagent Co., Ltd.). Cell lysates were sonicated to generate chromatin fragments of 200–300 bp (20 kHz; 4 pulses of 12 sec each, followed by 30 sec rest on ice between each pulse). Centrifugation was performed at 14,000 × g for 10 min at 4°C before protease inhibitor mixture II buffer (EMD Millipore) at a ratio of 9:1 and 60 µl protein G agarose was added and then cultured at 4°C for 1 h. After removing the agarose by centrifugation at 4,000 × g for 1 min at 4°C, antibody against TEAD1 (1:80; cat. no. ab133533; Abcam) or IgG (1:50; cat. no. ab6721; Abcam) was added into the supernatant. The precipitated DNA was purified using the ChIP DNA Clean & Concentrator kit (A&D Technology) according to the manufacturer's protocol. The precipitated chromatin DNA was recovered and assessed by qPCR.
7
Antibody Characterization for Protein Analysis
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The following antibodies were used in this study for western blot, immunohistochemistry, immunofluorescence staining and immunoprecipitation: YAP1 (Santa Cruz Biotechnology, sc-376,830, 1:100 dilution for western blot; 1:50 dilution for immunohistochemistry and 1:50 dilution for Immunofluorescence staining; Abcam, ab52771, 1:20 dilution for IP), p-YAP1 (Abcam, ab76252, 1:10000 dilution for western blot), α-SMA (Abcam, ab5694, 1:200 dilution for western blot; 1:100 dilution for immunohistochemistry and 1:100 dilution for immunofluorescence staining), FAP (Abcam, ab53066, 1:1000 dilution for western blot and 1:100 dilution for immunofluorescence staining), SRC (Signalway Antibody, #40790, 1:1000 dilution for western blot, 1:100 dilution for immunohistochemistry and 1:100 dilution for immunofluorescence staining), p-SRC (Abcam, ab4816, 1:1000 dilution for western blot), TEAD1 (Abcam, ab133533, 1:20 dilution for IP and 1:500 dilution for western blot), GAPDH (Sungene Biotech, KM9002, 1:5000 dilution for western blot).
8
Immunohistochemical Analysis of GC Markers
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Immunohistochemical staining was performed on 93 cases of GC and their paired adjacent tissues according to the standard method described previously using the following antibodies: anti-SCD1 (ab19862, 1:100, Abcam, Cambridge, UK), anti-YAP1 (14074S, 1:200, Cell Signaling Technology (CST), Danvers, MA, USA), anti-E-cadherin (14472S, 1:100, CST), anti-vimentin (5741S, 1:20, CST), anti-N-cadherin (13116S, 1:100, CST) and anti-TEA domain transcription factor 1 (TEAD1) (ab133533, 1:100, Abcam). Immunohistochemical (IHC) staining scores were evaluated based on the ratio and intensity of stained cells following the methods described in the previous study.12 (link) A categorisation of expression into three groups was based on the scores: negative, moderate and positive expression
9
Western Blot Analysis of Glycolytic Proteins
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Cells were lysed with RIPA lysis buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). Bicinchoninic acid (BCA) assay was used to quantify protein concentration. The proteins were separated on a gel prepared using PAGE Gel Fast Preparation Kit (Epizyme, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, USA). Membranes were blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), and then incubated with the following primary antibodies: anti-glucose transporter GLUT1 antibody (ab115730; Abcam, Cambridge, UK), anti-hexokinase II antibody (ab209847; Abcam), anti-TEF1/TEAD-1 antibody (ab133533; Abcam), anti-LDHA antibody (#3582; CST, Danvers, USA), anti-PKM2 antibody (#4053; CST), anti-N-cadherin antibody (#13116; CST), anti-Vimentin antibody (#5741; CST), anti-HIF-1α antibody (BF8002; Affinity Biosciences, Changzhou, China), anti-E-cadherin antibody (ab40772; Abcam) or mouse anti-β actin mAb (TA-09; ZSGB-BIO, Beijing, China). Subsequently, the membranes were incubated with the corresponding secondary antibody at room temperature for 1 h, and the blots were visualized using enhanced chemiluminescence (ECL) reagent.
10
Immunohistochemical Analysis of Tumor Markers
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Tumours resected from mice were fixed immediately, followed by paraffin embedding according to a standard protocol. For IHC staining, tumour sections were deparaffinized. Sodium citrate was used for antigen retrieval at 95 °C for 10 min. Then, 3% H2O2 was used to deplete endogenous peroxidase activity at 37 °C for 30 min. Next, the tumour tissues were blocked with 5% BSA, followed by incubation with anti-GPR4 (ab188931, Abcam), anti-YAP1 (14,074, Cell signalling), anti-TEAD1 (ab133533, Abcam), anti-Ki67 (#9449, Cell signalling), anti-Myc (ab32072, Abcam) or anti-Active RhoA -GTP (26,904, NewEast Biosciences) antibody. On the second day, the sections were washed three times with PBS and incubated with the corresponding HRP-linked secondary antibodies for 1 h at RT. IHC staining signals were presented with DAB Kit (Cell Signalling).
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