The treated cells were collected in RIPA buffer (Sigma, MO, USA). Nuclear and cytoplasmic protein extractions were prepared using NE-PER nuclear and cytoplasmic extraction kit (Thermo Scientific, MA, USA) following manufacturer’s instructions. Total protein content was measured by DC protein assay (Bio-Rad, CA, USA). The extracted proteins (20 μg) were then resolved on 10% SDS-PAGE and transferred to PVDF membrane. The membrane was then blocked with 5% skim milk or BSA, followed by overnight incubation with primary antibodies, anti-YAP/TAZ, anti-YAP (D8H1X), anti-Phospho-YAP1 (Ser127), anti-CCND1 and anti-GADPH (Cell Signaling, MA, USA), anti-GPER, anti-ERa and anti-Ki67 (Abcam, CA, USA), and anti-b-actin (Invitrogen, CA, USA) at 1:1000 dilution. After washing, the blots were incubated with goat anti-rabbit IgG (H + L)-HRP conjugate or goat anti-rabbit IgG (H + L)-HRP conjugate (Bio-rad, CA, USA). The protein bands were visualized with enhanced chemiluminescence reagent (Clarity Western ECL Substrate, Bio-Rad, CA, USA). GADPH and b-actin served as loading control.
Anti yap taz
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-YAP/TAZ is a laboratory reagent that can be used to detect and quantify the expression levels of the YAP and TAZ proteins in biological samples. YAP and TAZ are transcriptional co-activators that play key roles in the Hippo signaling pathway, which regulates cell growth, proliferation, and survival. Anti-YAP/TAZ can be used in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to investigate the involvement of YAP and TAZ in various cellular processes and disease states.
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Lab products found in correlation
Anti yap, by Cell Signaling Technology (5 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Anti lats1, by Cell Signaling Technology (2 mentions)
Anti p yap, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti phospho yap ser127, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti α tubulin, by Merck Group (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti egfr, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg h l hrp conjugate, by Bio-Rad (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti β actin, by Thermo Fisher Scientific (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
16 protocols using anti yap taz
1
Quantitative Western Blot Analysis of Protein Expression
2
Histological and Immunohistochemical Analysis of Mouse Intestinal Samples
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Mouse intestinal samples were collected, fixed overnight in 4% paraformaldehyde in PBS, embedded in paraffin, and sectioned at 5 µm. Sections were stained with hematoxylin–eosin for histological analysis. Immunohistochemistry on serial sections was performed according to the manufacturers’ protocols. Intraperitoneal injection of 30 mg/kg BrdU (Sigma) dissolved in 3 mg/mL 1× PBS was performed 2 h before tissue harvest. The primary antibodies used for immunohistochemistry were rabbit anti-β-catenin (1:500; Sigma), anti-YAP (1:100; Cell Signaling), anti-p-YAP (S127; 1:100; Cell Signaling), anti-YAP/TAZ (1:100; Cell Signaling), anti-Ki67 (1:1000; Novocastra), anti-Lats1 (1:100; Cell Signaling), and mouse anti-BrdU (1:50; Developmental Studies Hybridoma Bank). The signals were developed using the ABC kit purchased from Vector Laboratories according to the manufacturer's suggestions. Cy3-conjugated goat anti-rabbit and Alexa 488-conjugated goat anti-mouse secondary antibodies (Molecular Probes) were used for immunofluorescence.
3
Protein Analysis via SDS-PAGE and Western Blot
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Protein extraction, SDS-PAGE, and Western blot were performed as previously described [6 (link)]. Briefly, 40–60 μg of protein was separated by SDS-PAGE, electrophoretically transferred to PVDF membranes, blocked with 5% milk in TBS (50 mM Tris-HCl, pH 7.6; 150 mM NaCl), and probed overnight at 4 °C with the following antibodies: anti-YAP/TAZ (8418, Cell Signaling, Danvers, MA, USA), anti-Fibronectin (F3648, Sigma-Aldrich, St. Louis, MO, USA), and anti-GAPDH (sc-365062, Santa Cruz Biotechnology, Santa Cruz, CA, USA). To detect the primary antibody, horseradish-peroxidase-conjugated secondary antibodies and SuperSignal™ Luminol/Enhancer substrates (Thermo, Waltham, MA, USA) were used to generate chemiluminescence. Protein expression, measured as absolute pixel density, was determined using ImageJ software (v.1.53k, NIH, Bethesda, MD, USA).
4
Protein Detection in Cells and Spheroids
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In order to detect various proteins from cells and spheroids, a proper amount of samples were homogenized and digested in 1× RIPA buffer (Cell Signaling, Denver, MA, USA) which includes 1 mM PMSF (Fluka, Switzerland), 2 g/mL Aprotinin (Sigama, Steinheim, Germany), 1 mM DTT(Invitrogen, Carlsbad, CA, USA) and phosphatase inhibitor cocktail solution (GenDEPOT, Barker, TX, USA) on ice for 1 hr. After the digestion, samples were centrifuged for 25 min at 14,000 rpm in cold microcentrifuge. Then supernatants were collected for use. Western blot experiments were performed following the standard protocol. The following primary antibodies were purchased: anti-YAP/TAZ (8418, Cell Signaling) and anti-EpCAM (ab71916, Abcam). anti-EpCAM was used and for internal control, an anti-GAPDH antibody (2118, Cell Signaling) was used. Anti-rabbit IgG-HRP (A0545, Sigma) was used as the secondary antibody. Specific bands were detected using the enhanced chemiluminescence (ECL) Western blot detection system (Amersham Pharmacia Biotech, Piscataway, NJ, USA).
5
Western Blotting of PTEN and Akt Pathway
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Western blotting was performed according to standard method described previously [25 (link)] using the following antibodies: anti-PTEN (#9559; 1:1000), anti-phosphorylated (p)-PTEN (Ser380/Thr382/383) (#9554; 1:1000), anti-Akt (#4691; 1:1000), anti-p-Akt (Ser473) (#4060; 1:1000), anti-p-Akt (Thr308) (#13088; 1:1000), anti-Bad (#9239; 1:1000), anti-p-Bad (Ser136) (#4366; 1:1000), anti-p-FoxO1 (Ser256) (#9461; 1:1000), anti-FoxO1(#2880; 1:1000), anti-p27 Kip1 (#3686; 1:1000), anti-p-GSK-3β (#9323; 1:1000), anti-GSK-3β (#9315; 1:1000), anti-p-YAP (Ser 127) (#13008; 1:1000), anti-YAP/TAZ(#8418; 1:1000), anti-SAV1 (#13301; 1:1000), anti-LATS1 (#3477; 1:1000), anti-LATS2 (#5888; 1:1000), anti-MOB1 (#8699; 1:1000), anti-histone H3 (#4499; 1:1000), and anti-TEAD (#13295; 1:1000) from Cell Signaling Technology (Danvers, MA, USA) and anti-GAPDH (BA2913, 1:1000) from Boster Biological Technology (Preston, CA, USA).
6
Histological Analysis of Mouse Skin
7
Antibody Validation for Immunoblotting
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The following antibodies were used in this study (with dilution factor for immunoblotting): anti-PKN2 (#2612, 1:1,000), anti-YAP (#14074, 1:1,000), anti-YAP/TAZ (#8418, 1:1,000), anti-phospho-YAP (Ser127) (#4911, 1:1,000), anti-phospho-EGFR (Tyr1068) (#3777, 1:1,000), anti-EGFR (#4267, 1:20,000), anti-phospho-AKT (Ser473) (#4058, 1:1,000), anti-AKT (#9272, 1:5,000), anti-phospho-ERK1/2 (Thr202/Tyr204) (#9101, 1:1,000), anti-ERK1/2 (#9102, 1:5,000), anti-ARIH2/TRIAD1 (#13689, 1:1,000), anti-BTAF1 (#2637, 1:1,000), anti-GNAQ (#14373, 1:1,000), anti-HSP90 (#4877, 1:5,000), anti-ALDOA (#8060, 1:5,000), anti-METAP2 (#12547, 1:1,000), anti-GAPDH (#2118, 1:5,000), anti-RHOA (#2117, 1:1,000), anti-Cofilin (#5175, 1:1,000), anti-phospho-Cofilin (Ser3) (#3313, 1:1,000), from Cell Signaling Technology; anti-α-Tubulin (T6074, 1:20,000) and anti-β-Actin (A1978, 1:20,000) from Sigma; anti-GNB2 (ab81272, 1:1,000), anti-RIC8A (ab97808, 1:1,000), anti-USP22 (ab195289, 1:1,000), anti-CUL5 (ab184177, 1:1,000), anti-RNF7 (ab181986, 1:1,000), anti-PDCD10 (ab180706, 1:1,000), from Abcam; anti-KCTD5 (#15553–1-AP, 1:1,000), anti-PSAT1 (#10501–1-AP, 1:5,000), from Proteintech; Goat Anti-Rabbit IgG Antibody, (H+L) HRP conjugate (#AP307P, 1:5,000), Goat Anti-Mouse IgG Antibody, (H+L) HRP conjugate (#AP308P, 1:5,000), from Millipore Sigma.
8
Immunofluorescence Staining of Cell Markers
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Cells were fixed with 4% paraformaldehyde for 15 min, washed three times for 5 min each with PBS, and incubated for 1 h at room temperature in blocking buffer consisting of PBS, 0.3% Triton X-100 (Sigma Aldrich), and 5% normal goat serum (Sigma Aldrich). Samples were then incubated overnight at 4°C in antibody dilution buffer consisting of PBS, 0.3% Triton X-100, and 2% bovine serum albumin (Sigma Aldrich) containing primary antibodies. The following antibodies were used: anti-EGFR (Santa Cruz Biotechnology sc-101), anti-E-cadherin (Cell Signaling 3195 or Thermo Fisher 13-1900), anti-YAP/TAZ (Cell Signaling 8418). The next day, samples were washed three times for 5 min each with PBS and incubated for 1 h at room temperature in antibody dilution buffer containing Alexa Fluor-conjugated secondary antibodies (Invitrogen). Samples were then washed three times for 5 min each with PBS, incubated for 5 min at room temperature in PBS containing Hoechst 33342 (Invitrogen), and washed twice with PBS before imaging under confocal microscopy.
9
Huaier Modulates Cellular Signaling
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Cells were treated with a series concentrations of Huaier (0, 5, 10 and 15mg/ml) for 24h, and then lysed by cell lysis buffer for Western and IP (Beyotime, P0013). Protein concentration was detected with BCA Protein Assay Kit (KeyGen BioTECH, KGP902). After that, 20µg proteins were separated by 10% or 12% SDS-PAGE and transferred onto Pure Nitrocellulose Blotting Membrane (Pall Corporation, P/N 66485). Then, the blots were blocked for nonspecific binding with 5% non-fat milk in PBST (PBS, Tween-20, pH7.4) at room temperature for 1h. Next, the blots were incubated overnight at 4℃ with 5% non-fat milk containing primary antibodies which are listed as follows: anti-PCNA (ImmunoWay, YM3031), anti-Ki-67 (ImmunoWay, YT2467), anti-β-actin (ImmunoWay, YM3028), anti-Bcl-2 (ImmunoWay, YM3041), anti-Bax (ImmunoWay, YT0455), anti-E-cadherin (ImmunoWay, YT1454), anti-N-cadherin (ImmunoWay, YT2988), anti-YAP1 (abcam, ab52771), anti-p-YAP1 (ab76252), anti-CyclinD1 (abcam, ab134175), anti-Cleaved Caspase Substrate Motif (Cell Signaling, #8698) and anti-YAP/TAZ (Cell Signaling, #8418). After that, incubating blots with secondary antibodies conjugated with HRP (Cell Signaling, #7074 or #7076) for 1h at room temperature. Finally, after washing three times with PBST, the blots were visualized by New Super ECL Assay (KeyGen BioTECH, KGP1128).
10
Investigating Signaling Pathways in Cancer
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Dasatinib, BIBW2992, PD0325091 and Etoposide were purchased from Selleck Chemicals (Houston, TX) and prepared as 20 mM stock solutions in DMSO. Antibodies used included anti-YAP-TAZ, pLats, Lats1, Lats2, pMST1/2, pH2AX, MEK, pMEK(S217/221), ERK, pERK(T202/Y204), pRb, pS6, LC3A/B, ATM, pChk1, Chk1, pChk2, Chk2, pCDC25c, pATRIP, p27, p21, pp38, β-catenin, cyclin D1, survivin, p4EBP1, pSMAD2(S465–467), p70S6K, pCDC2, CDC6, γH2AX, ATR, pDNA-PK(S2056) and DNA-PK, (Cell Signaling Technology, Danvers, MA); BRAF and cyclin E (Santa Cruz, Dallas, TX) and ATM, Flag M2 and β-actin (Sigma-Aldrich, St. Louis, MO); pATM(S1981) (GeneTex, Irvine, CA). Predesigned siRNAs of the target genes were purchased from Dharmacon (Pittsburgh, PA) or Thermo Scientific (Rodckford, IL). pcDNA4-Chk1-Flag and pCMV5-TOPO-3Xflag-TAZ plasmids were purchased from Addgene (Cambridge, MA). WTBRAF plasmid was provided by Dr. W. Kolch (Systems Biology Irland and The Conway Institute, University College Dublin).
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