IF and IHC were performed according to the conventional protocols. The primary antibodies used for IF were anti-YAP (Abcam, Hong Kong, China #ab52771) and anti-βTrCP (Abcam, #ab233638). The primary antibodies used for IHC were: anti-YAP (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-101199) and anti-ISG15 (Abcam, #ab233071). For IB, the proteins were resolved on SDS-PAGE gels according to the conventional protocols. The primary antibodies used were anti-ISG15 (Abcam, #ab233071), anti-YAP (Abcam, #ab52771 and Santa Cruz, #sc-101199), anti-GAPDH (CST, #5174 and #51332), anti-Ub (Abcam, #ab7780 and #ab7254), anti-PSMB5 (Abcam, #ab167341), anti-βTrCP (Abcam, #ab71753 and #ab233638), anti-YAPO241 (developed by Biolynx, Hangzhou, China), anti-YAPP127 (Abcam, #ab76252), anti-YAPP397 (CST, Boston, MA, USA, #13619), anti-HA (Abcam, #ab9110 and #ab18181), anti-TEAD4 (Abcam, #ab197589 and #ab58310), anti-6PGL (Abcam, #ab229872), anti-FLAG (CST, #8146 and #2368), anti-UbCH8 (Abcam, #ab177485), anti-HERC5 (Invitrogen, Carbsland, CA, USA, #703675), anti-ATG5 (Abcam, #ab221604), anti-LATS1 (Abcam, #ab243656), anti-CK1 (Abcam, #ab270997 and #ab115293), anti-SMAD2 (Abcam, #ab40855), anti-alpha fetoprotein (AFP, Abcam, #ab284388) and anti-albumin (Alb, Abcam, #ab207327). ELISA kits (Yingxin, Shanghai, China) were used to measure the concentration of YAP protein and Rib-5-P.
Anti yap
Manufactured by Abcam
Sourced in United States, China, Hong Kong, United Kingdom
Anti-YAP is a laboratory product that recognizes the YAP protein. YAP is a transcriptional regulator involved in the Hippo signaling pathway, which plays a role in cell growth and survival. Anti-YAP can be used to detect and study the YAP protein in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti lats1, by Abcam (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Anti cd63, by Abcam (2 mentions)
Anti bax, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti ki67, by Abcam (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Anti col1, by Abcam (2 mentions)
Citrate buffer, by Vector Laboratories (1 mentions)
Anti β catenin, by Merck Group (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Ab205270, by Abcam (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Anti ha, by Abcam (1 mentions)
Anti smad2, by Abcam (1 mentions)
22 protocols using anti yap
1
Comprehensive Protein Analysis Protocol
2
Immunohistochemical Analysis of Cancer Biomarkers
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Formalin-fixed, paraffin-embedded sections were obtained from archival human tubular adenomas, pancreatic acinar cell carcinomas, ovarian serous carcinomas, and pancreatic ductal adenocarcinomas that had been surgically removed at the Johns Hopkins Hospital. The diagnosis of these cancers was rendered based on established criteria by specialized pathologists (A. Maitra and R.A. Anders). Heat-induced antigen retrieval was performed in a steamer using citrate buffer (pH 6.0; Vector Laboratories) for 25 min followed by 30 min of cooling. Nonspecific binding was blocked for 30 min with 5% goat serum in TBST. Serial sections were then incubated with two primary antibodies—anti-YAP (1:150; Epitomics) and anti-β-catenin (1:500; Sigma)—overnight at 4°C. The signals were developed using the ABC kit purchased from Vector Laboratories according to the manufacturer's suggestions. Finally, sections were counterstained with Harris hematoxylin, subsequently rehydrated in distilled H2O and graded series of ethanol (70%, 95%, and 100%), and mounted.
3
Immunohistochemical Analysis of ITCH and YAP
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Paraffin-embedded lung tissue sections obtained from above were deparaffinized with xylene and rehydrated with step-down changes of ethanol. Antigen retrieval was performed in pre-heated 10 mM sodium citrate buffer pH 6.0 using pressurized for 15 minutes. Endogenous peroxidase was blocked with 3% H2O2 for 10 minutes. The sections were then incubated with blocking solution for 30 minutes to reduce non-specific binding followed by incubation with the primary antibody (monoclonal anti-ITCH antibody (Sigma Aldrich, St. Louis., MO, USA) [dilution of 1:100], and anti-YAP (Epitomics, Burlingame, CA, USA) [dilution of 1:200]), at 4°C in a humidified chamber for overnight incubation. Post-washing, slides were subsequently incubated with horseradish peroxidase-conjugated antibody for 30 minutes. Detected was done using a freshly prepared 3, 3 diamminobenzidine tetrahydrochloride using DAKO Liquid DAB Substrate-Chromogen (Dako Corporation, Carpinteria, California, USA) solution for several minutes at room temperature.
4
Protein Extraction and Western Blot Analysis
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OE tissues were lysed in lysis buffer for 30 min on ice. The lysis buffer consisted of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% Triton X-100, 5 mM sodium fluoride, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 2 mM sodium orthovanadate, and a protease inhibitor cocktail (P8340, Sigma). The lysates were then centrifuged at 12,000 rpm for 30 min to obtain the suspended proteins. The proteins were extracted after adding 5× loading buffer and denatured at 100°C for 10 min before being separated by SDS-PAGE (8%–12%). The proteins were then transferred into nitrocellulose membranes (Life Sciences) and immersed in 5% skim milk for blocking at room temperature for 1 h, followed by an overnight incubation with primary antibodies at 4°C, then rinsed with Tris-buffered saline with Tween 20 and incubated for 1 h at room temperature after adding the appropriate secondary antibodies (1:5,000, Bio-Rad). Primary antibodies included anti-p-YAP (Ser127, 1:1,000, #4911, CST), anti-YAP (1:1,000, ab205270, Abcam), anti-TAZ (1:1,000, DF4653, Affinity), anti-MOB1 (1:1,000, #13730, CST), and anti-p-MOB1 (Thr35, 1:1,000, #8699, CST). Anti-GAPDH (1:5,000, #2118, CST) and anti-β-actin (1:10,000, A5316, Sigma) were used as loading controls. Protein bands were detected through ECL (Bio-Rad), and analyzed by Quantity One software (Bio-Rad).
5
Immunofluorescence Staining of YAP in Cells
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Cells were fixed with 3.4% paraformaldehyde (PFA) (Maokang, Shanghai, China) for 20 min and then permeabilized with 0.5% Triton X-100 (Sigma-Aldrich). After blocking in 3% PBS-BSA for 30 min, slides were incubated with anti-YAP (1:200, Abcam, CA, USA). After washing with PBS, slides were incubated with goat anti-mouse FITC conjugated secondary antibodies for 1 h at 37°C. The nuclei were stained using DAPI. The targeted proteins were detected using confocal microscopy (ZEISS LSM700) and a laser-scanning confocal microscope image system. In addition, the immunofluorescence staining of pulmonary tissues was performed as previously described.19 (link)
6
Protein Extraction and Western Blot Analysis
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Total protein was extracted by RIPA and phenylmethane sulfonyl fluoride (PMSF) lysate buffers. The protein concentrations were determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). The procedure was performed as previously described [18 (link)]. The blots were incubated with primary antibodies, including anti-YAP (Abcam, 1 : 1000 dilution), anti-p-YAP Ser127 (Abcam, 1 : 2000 dilution), anti-cyclin D (Santa Cruz Biotechnology, 1 : 1000 dilution), or anti-β-actin (1 : 2000, Proteintech, Wuhan, China), at 4°C overnight. The membranes were then incubated with secondary antibodies for 2 h. The blots were visualized using an enhanced chemiluminescence substrate kit (Thermo Fisher Scientific, Rockford, USA), and the results were analyzed with Image J software.
7
Extracellular Vesicle Protein Profiling in Cardiac and Mesenchymal Cells
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Total protein was extracted from H9c2 cells and MSCs-exosomes by
radio-immunoprecipitation assay (RIPA) lysis buffer (TaKaRa, Japan) with
protease inhibitors (Roche, China). The concentrations of proteins were detected
by a bicinchoninic acid (BCA) protein assay kit (Pierce, Netherlands). Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
separate equal quantities of total proteins (20 μg per lane), and then separated
proteins were transferred onto polyvinylidene difluoride membrane (Millipore,
MA, USA). Membranes were blocked by PBS-5% fat-free dried milk at room
temperature for 1 h and then incubated at 4 ℃ overnight with anti-CD63
(1:1,000), anti-YAP (1:2,000), anti-phosphor (p)-YAP (1:2,000), anti-tafazzin
(TAZ) (1:2,000), anti-caspase 3 (1:1,000), anti-B-cell lymphoma-2 (Bcl-2)
(1:2,000), anti-Bax (1:1,000), and anti- glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (1:3,000) (Abcam, UK). Then, the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody was incubated with
membranes for 2 h. Protein bands were visualized by ChemiDoc XRS+ system
(Bio-Rad, CA, USA).
radio-immunoprecipitation assay (RIPA) lysis buffer (TaKaRa, Japan) with
protease inhibitors (Roche, China). The concentrations of proteins were detected
by a bicinchoninic acid (BCA) protein assay kit (Pierce, Netherlands). Sodium
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to
separate equal quantities of total proteins (20 μg per lane), and then separated
proteins were transferred onto polyvinylidene difluoride membrane (Millipore,
MA, USA). Membranes were blocked by PBS-5% fat-free dried milk at room
temperature for 1 h and then incubated at 4 ℃ overnight with anti-CD63
(1:1,000), anti-YAP (1:2,000), anti-phosphor (p)-YAP (1:2,000), anti-tafazzin
(TAZ) (1:2,000), anti-caspase 3 (1:1,000), anti-B-cell lymphoma-2 (Bcl-2)
(1:2,000), anti-Bax (1:1,000), and anti- glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (1:3,000) (Abcam, UK). Then, the goat anti-rabbit
horseradish peroxidase-conjugated secondary antibody was incubated with
membranes for 2 h. Protein bands were visualized by ChemiDoc XRS+ system
(Bio-Rad, CA, USA).
8
Western Blot Analysis of LATS1, YAP, and p-YAP
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Total protein was extracted from A375 and MV3 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quantified using a BCA assay kit (Beyotime Institute of Biotechnology) and 20 µg protein/lane was separated via 10% SDS-PAGE. The separated proteins were subsequently transferred onto PVDF membranes and blocked with 5% non-fat milk for 2 h at room temperature. The membranes were then incubated with the following primary antibodies at 4°C overnight: Anti-LATS1 (1:1,000; cat. no. ab243656; Abcam), anti-phosphorylated (p)-Yes 1 associated transcriptional regulator (YAP; 1:1,000; cat. no. ab76252; Abcam), anti-YAP (1:1,000; cat. no. ab52771; Abcam) and anti-β-actin (1:1,000; cat. no. ab8226; Abcam). Following the primary antibody incubation, the membranes were incubated with HRP-conjugated anti-mouse (1:10,000; cat. no. ab6289; Abcam) or anti-rabbit (1:10,000; cat. no. ab6721; Abcam) secondary antibodies for 2 h at room temperature. Protein bands were visualized using an ECL reagent (Pierce; Thermo Fisher Scientific, Inc.) and densitometric analysis was performed using ImageJ software (version 1.8.0; National Institutes of Health). β-actin was used as the internal loading control.
9
Western Blot Analysis of Protein Expression
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WB was conducted according to conventional protocols. Briefly, cells treated as indicated were harvested and lysed using RIPA buffer (Beyotime, China). Proteins were extracted and transferred to nitrocellulose membranes via SDS–PAGE. Then, membranes were blocked with 5% skimmed milk for 1 h at room temperature prior to incubation with primary antibodies overnight at 4 °C. The primary antibodies used were anti-O-GlcNAc (Abcam, #ab2735), anti-OGT (Abcam, #ab184198 or #ab177941), anti-YAP (Abcam, #ab52771), anti-β-Tubulin (CST, #2128), anti-Histone-H3 (Santa Cruz, #sc-10809), anti-TFRC (Abcam, #ab84036), anti-SLC7A11 (Abcam, #175186), and anti-GAPDH (CST, #5176). For nuclear and cytosolic separation, nuclear extraction was conducted according to the manufacturer’s protocols for the Nuclear Extraction Kit (Active Motif, USA). Membranes were incubated with HRP-linked secondary antibodies [anti-rabbit (CST, #7074) or anti-mouse (CST, #7076)] for 1 h at room temperature. Signals were detected using Pierce™ ECL Western Blotting Substrate (Thermo Scientific, USA).
10
Western Blot Analysis of Key Signaling Proteins
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Cells were harvested, washed in cold PBS, and lysed in Western/IP lysis buffer (Beyotime, Shanghai, China). The whole-cell lysates were centrifuged at 12,000 rpm for 15 min at 4 °C. Protein concentrations were measured using the Bradford method. Proteins were separated via SDS-PAGE, and transferred onto a nitrocellulose membrane. The membranes were blocked using 5% evaporated milk and 1% Tween-20 in PBS for 1 h and were incubated with primary antibodies dissolved in PBS containing 1% Tween-20 overnight at 4 °C. The primary antibodies were anti-YAP (Abcam, Hong Kong, China, #ab52771), anti-β-catenin (Abcam, #ab32572), anti-TGF-β (Abcam, #ab31013), anti-STAT3 (Abcam, #ab68153), anti-HNF4a (Abcam, #ab41898), anti-c-Myc (Abcam, #ab32072), anti-FOXO1 (Abcam, #ab39670) and anti-GAPDH (Cell Signaling Technology (CST), Boston, MA, USA, #5174). On the second day, the blots were incubated with the appropriate secondary antibody: IRDye 800CW goat anti-mouse IgG (LICOR, Lincoln, NE, USA, #926-32210), IRDye 800CW goat anti-rabbit IgG (LICOR, #926-32211), anti-rabbit IgG, HRP-linked antibody (CST, #7074) or anti-mouse IgG, HRP-linked antibody (CST, #7076). The signals were detected using the Odyssey two-color infrared laser imaging system (LICOR) or HRP-based chemiluminescence analysis.
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