The homogenate of lung tissues, pulmonary arteries, and PASMCs were prepared and subjected to electrophoresis on sodium-dodecyl-sulfate polyacrylamide gels, and then transferred onto polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk or 5% bovine serum albumin, and incubated with monoclonal antibody including anti-Cyr61 (Abcam, USA), anti-AKT (Cell signaling Technology, USA), anti-p-AKT (Cell signaling Technology, USA), anti β-actin (Sigma-Aldrich, St Louis, MO, USA) or anti-GAPDH (ZSGB-BIO, China) at 4℃ overnight, followed by the matched secondary antibody (Protein Tech, Chicago, IL, USA). Immunopositive spots were probed using ECL-PlusTM (Amersham Biosciences, Chalfont St Giles, UK).
Anti cyr61
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-CYR61 is a laboratory reagent used for the detection and quantification of the CYR61 protein. CYR61 is a secreted, heparin-binding, and cysteine-rich protein that regulates various cellular processes. Anti-CYR61 can be used in techniques such as Western blotting, immunohistochemistry, and ELISA to study the expression and localization of CYR61 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Sirna cyr61, by Sangon (2 mentions)
Anti ctgf, by Abcam (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Ecl plu, by Cytiva (1 mentions)
Matched secondary antibody, by Proteintech (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti gapdh, by ZSGB-BIO (1 mentions)
Pierce co immunoprecipitation co ip kit, by Thermo Fisher Scientific (1 mentions)
Anti itgav, by Abcam (1 mentions)
Anti itgb1, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Anti lgr5, by Abcam (1 mentions)
Anti sox9, by Abcam (1 mentions)
Anti flag, by Proteintech (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti β actin, by Bioworld Technology (1 mentions)
Anti ck19, by Abcam (1 mentions)
Anti myc, by Proteintech (1 mentions)
Protease inhibitors and phosphatase inhibitors, by Beyotime (1 mentions)
7 protocols using anti cyr61
1
Western Blot Analysis of Lung Tissue
2
Cyr61 Protein Interaction Analysis
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Anti-Cyr61, Anti-ITGAV, Anti-ITGB1, and goat Anti-Rabbit lgG H&L were from Abcam. Human recombinant Cyr61 was obtained from GeneTex. SiRNA-Cyr61 and negative control siRNA were purchased from Sangon Biotech (Shanghai). Pierce Co-Immunoprecipitation (Co-IP) Kit (26149) was purchased from Thermo scientific. The recombinant plasmid of pcDNA3.1-Cyr61 was constructed by our lab.
3
Comprehensive Protein Analysis Protocol
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Tissues or cultured cells were lysed with RIPA lysis buffer with protease inhibitors and phosphatase inhibitors (Beyotime, Shanghai, China) using standard methods. After centrifugation and quantification, 25 µg of protein was loaded for blotting with the indicated specific antibodies. A NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo Fisher Scientific, Waltham, USA) was used to isolate nuclear and cytoplasmic fractions according to the manufacturer’s protocol. The following antibodies were used: anti-Sox9 (1:1000, Abcam, Cambridge, UK), anti-HNF4α (1:2000, Abcam, Cambridge, UK), anti-TERT (1:1000, Novus, Colorado, USA), anti-E-Cadherin (1:1000, Abcam, Cambridge, UK), anti-Vimentin (1:1000, CST, Danvers, USA), anti-CK19 (1:1000, Abcam, Cambridge, UK), anti-LGR5 (1:1000, Abcam, Cambridge, UK), anti-Epcam (1:1000, Abcam, Cambridge, UK), anti-P65 (1:1000, CST, Danvers, USA), anti-p-P65 (1:1000, CST, Danvers, USA), anti-Bcl3 (1:100, Abcam, Cambridge, UK), anti-Histone H3 (1:1000, Proteintech, Rosemont, USA), anti-YAP1 (1:1000, CST, Danvers, USA), anti-CTGF (1:1000, Abcam, Cambridge, UK), anti-Cyr61 (1:1000, Abcam, Cambridge, UK), anti-His (1:5000, Proteintech, Rosemont, USA), anti-Flag (1:2000, Proteintech, Rosemont, USA), anti-Myc (1:1000, Proteintech, Rosemont, USA), anti-Ubiquitin (1:1000, CST, Danvers, USA), and anti-β-actin (1:4000, Bioworld, MN, USA).
4
IGF-1 Signaling Pathway Regulation
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Recombinant CYR61 and SP600125 were purchased from Sigma-Aldrich (Lyon, France), recombinant IGF-1 was purchased from R&D Systems (Lille, France).
Neutralizing anti-IGF1 antibody was purchased from R&D Systems. Rabbit polyclonal anti-CYR61 and anti-pIGF1Rβ were purchased from Abcam (Cambridge, UK), anti-Actin was purchased from Sigma Aldrich and anti-IGF1Rβ, anti-pGSK3β, anti-pJNK, anti-JNK were purchased from Cell Signaling Technology (Saint Quentin en Yvelines, France). Mouse monoclonal anti-E-cadherin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti- GSK3β was purchased from Cell Signaling Technology, and anti-IGF1 was purchased from R&D Systems.
Neutralizing anti-IGF1 antibody was purchased from R&D Systems. Rabbit polyclonal anti-CYR61 and anti-pIGF1Rβ were purchased from Abcam (Cambridge, UK), anti-Actin was purchased from Sigma Aldrich and anti-IGF1Rβ, anti-pGSK3β, anti-pJNK, anti-JNK were purchased from Cell Signaling Technology (Saint Quentin en Yvelines, France). Mouse monoclonal anti-E-cadherin was purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA), anti- GSK3β was purchased from Cell Signaling Technology, and anti-IGF1 was purchased from R&D Systems.
5
Evaluating YAP and Annexin V Protein Levels in NCTD and DDP-Treated A549/DDP Cells
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For the analysis of the protein levels of YAP and Annexin V, the A549/DDP cells were grown on coverslips in a 24-well plate overnight and after 24 h, they were treated with NCTD (8 µg/ml), DDP (6 µg/ml) or co-treated of NCTD (8 µg/ml) and DDP (6 µg/ml). After 48 h, the cells were fixed in 4% formaldehyde for 30 min and blocked in 3% BSA in PBS for 30 min. The coverslips were subsequently incubated with rabbit anti-YAP (#8418; Cell Signaling Technology, Danvers, MA, USA), anti-CYR61 (24448; Abcam, Cambridge, MA, USA), anti-CTGF (6992; Abcam) and anti-Annexin V (sc-32321; Santa Cruz Biotechnology) monoclonal antibodies at a 1:1,000 dilution in PBS containing 3% BSA. Alex Fluor AF 488 (green, 1:500, A-11029; Invitrogen, Carlsbad, CA, USA) and 594 (red, 1:500, A-11032; Invitrogen) anti-rabbit monoclonal secondary fluorescence antibodies at a 1:1,000 dilution in PBS containing 3% BSA. In addition, 3 µg/ml Hoechst (cat. no. E607328; Sangon Biotech Co., Ltd.) was used for nuclear staining at 25°C for 30 min. Images were obtained with Zeiss Axio Imager Z1 fluorescence microscope.
6
Exploring CYR61 Regulation in Breast Cancer
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SiRNA-CYR61 and negative SiRNA-control were purchased from Sangon Biotech (Shanghai). Pierce S-Nitrosylation western blot kit, immobilized anti-TMT Resin and TMT elution buffer were purchased from Thermo scienti c. Anti-CYR61 was from Abcam. The recombinant plasmid of pcDNA3.1-CYR61 was constructed by our lab.
Cell lines and culture MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were incubated with Leibovitz's L-15 medium (Catalog No. containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in a free gas exchange with atmospheric air. Human pulmonary microvascular endothelial cells (HPMEC) were purchased from Promocell, and were cultured in ECM with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in 5% CO 2 atmosphere.
Cell lines and culture MDA-MB-231 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA), and were incubated with Leibovitz's L-15 medium (Catalog No. containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in a free gas exchange with atmospheric air. Human pulmonary microvascular endothelial cells (HPMEC) were purchased from Promocell, and were cultured in ECM with 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin at 37℃ in 5% CO 2 atmosphere.
7
Western Blot Analysis of CYR61, SREBP-2, and Cadherins
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Cells were lysed in cold RIPA lysis buffer (Beyotime, Shanghai, China) and proteins in the supernatants were quantified. After separation by 10% SDS-PAGE, proteins were transferred onto polyvinylidene difluoride membranes (Life Technologies, Carlsbad, CA, USA). The blots were blocked with 10% nonfat milk in PBS and then probed with anti-CYR61 (Abcam, Cambridge, MA, USA), anti-SREBP-2 (Santa Cruz, Dallas, TX, USA), anti-E-cadherin, anti-N-cadherin or anti-β-actin antibody (all from Cell Signaling Technology, Inc., Danvers, MA, USA) for 1 h at room temperature. After washes, the blots were incubated with HRP-linked secondary antibodies and observed using an enhanced chemiluminescence kit (Pierce Chemical, Rockford, IL, USA). Protein levels were normalized to β-actin.
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