Cyr61

Manufactured by Santa Cruz Biotechnology

Sourced in United States

CYR61 is a protein that functions as a secreted, heparin-binding, extracellular matrix-associated protein. It is involved in cell adhesion, migration, proliferation, and differentiation.

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Lab products found in correlation

Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions) Survivin, by Cell Signaling Technology (2 mentions) C myc, by Cell Signaling Technology (2 mentions) Verteporfin, by Novartis (2 mentions) Vegfa, by Santa Cruz Biotechnology (2 mentions) Pakt s473, by Cell Signaling Technology (2 mentions) Nitrocellulose membrane, by Merck Group (2 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Flag tag (flag), by Merck Group (1 mentions) Bromodeoxyuridine (brdu), by BD (1 mentions) Occludin, by Thermo Fisher Scientific (1 mentions) Gapdh, by Abcam (1 mentions) P lats1, by Cell Signaling Technology (1 mentions) Ubiquitin, by BD (1 mentions) Lats2, by Cell Signaling Technology (1 mentions) Lats1, by Fortis Life Sciences (1 mentions) Pals1, by Santa Cruz Biotechnology (1 mentions) Yap p s127, by Cell Signaling Technology (1 mentions) γ tubulin, by Santa Cruz Biotechnology (1 mentions) Lamin b, by Santa Cruz Biotechnology (1 mentions)

Spelling variants of this product

Anti-CYR61 (5 citations) Anti-Cyr61 (sc-13100) (4 citations) CYR61 antibody (3 citations)

12 protocols using cyr61

Antibody-based Western Blot and Immunofluorescence

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The indicated antibodies against the following proteins/peptides were used for Western blot analyses: Flag (F3165; Sigma-Aldrich), HA (MMS-101P; Covance), Myc (sc-40; Santa Cruz Biotechnology), GFP (sc-9996; Santa Cruz Biotechnology), WWP1 (H00011059-M01; Novus Biologicals), WWP2 (A302-935A; Bethyl Laboratories), AMOTL2 (sc-82501; Santa Cruz Biotechnology), USP9X (Abnova, H00008239-M01), CRB3 (NBP1-81185; Novus Biologicals), PALS1 (Santa Cruz, sc-365411), LATS1 (A300-477A; Bethyl Laboratories), LATS2 (#5888; Cell Signaling Technology), p-LATS1 (#8654; Cell Signaling Technology), YAP p-S127 (#4911; Cell Signaling Technology), YAP (H00010413-M01; Novus Biologicals), TAZ (#4883S; Cell Signaling Technology), CTGF (sc-14939; Santa Cruz Biotechnology), CYR61 (sc-13100; Santa Cruz Biotechnology), ubiquitin (550944; BD Biosciences), γ-tubulin (sc-7396; Santa Cruz Biotechnology), occludin (71-1500; Invitrogen), lamin B (sc-6217; Santa Cruz Biotechnology), and GAPDH (ab125247; Abcam); rabbit anti-AMOTL2 p-S159 was a kind gift from Dr Wanjin Hong (National University of Singapore). Antibodies used for immunofluorescence analyses were: YAP (H00010413-M01; same as for Western blot; Novus Biologicals), GFP (ab13970; Abcam), and BrdU (555627; BD Biosciences). The antibodies used for immunoprecipitations were the same as those used for Western blot analyses.

Hwang D., Kim M., Kim S., Kwon M.R., Kang Y.S., Kim D., Kang H.C, & Lim D.S. (2021). AMOTL2 mono-ubiquitination by WWP1 promotes contact inhibition by facilitating LATS activation. Life Science Alliance, 4(10), e202000953.

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Verteporfin and Metformin Cytotoxicity Protocol

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Verteporfin (Visudyne) was obtained from Novartis (Novartis, Basel, Switzerland).
Metformin, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and ribonuclease-A were purchased from Sigma Aldrich (St.Louis, MO, USA). Propidium iodide was purchased from Invitrogen (Carlsbad, CA, USA). The following primary antibodies were purchased from Cell Signaling technology (Danvers, MA, USA) and used diluted 1:1000 unless stated otherwise: c-myc, axl, phospho-S6 ribosomal protein (Ser235/236), phospho-4EBP1 (Thr37/46), p21 Waf1/Cip1, p27Kip1, LC3B, phospho-p38 MAPK (Thr180/Tyr182) and p44/42 MAPK (Erk1/2), PCNA (1:2000), 4-Oct, survivin, pAkt (S473) (1:2000), pAkt (T308). The following primary antibodies were purchased from Epitomics (Burlingame, CA, USA): cyclin D1, D3, E1, E2, A2 and the following from Santa Cruz (Dallas, Texas, USA): cyr61 (1:500), VEGFA (1:500), CTGF (1:500).

Brodowska K., Moujahed A., Marmalidou A., zu Horste M.M., Cichy J., Miller J.W., Gragoudas E, & Vavvas D.G. (2014). The clinically used photosensitizer Verteporfin (VP) inhibits YAP-TEAD and human retinoblastoma cell growth in vitro without light activation. Experimental eye research, 124, 67-73.

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Protein Expression Analysis Protocol

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The cells were lysed in radioimmunoprecipitation lysis buffer (Beyotime), and the protein concentrations were determined. Approximately 60 μg of protein was separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The primary antibodies used were as follows: YAP (sc-15407; Santa Cruz Biotechnology), Cyr61 (sc-13100; Santa Cruz Biotechnology), CTGF (sc-101586; Santa Cruz Biotechnology), CCND1 (sc-20044; Santa Cruz Biotechnology), LC3B and Beclin1 (3868S and 3738, respectively; Cell Signaling Technology), cleaved PARP and caspase 3 (9541 and 9661, respectively; Cell Signaling Technology), and Atg-3 and -5 (3415 and 2630, respectively; Cell Signaling Technology).

Xiao L., Shi X.Y., Zhang Y., Zhu Y., Zhu L., Tian W., Zhu B.K, & Wei Z.L. (2016). YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells. OncoTargets and therapy, 9, 1105-1114.

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Western Blot Analysis of Hypoxic Signaling

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Western blot analysis was performed as described previously (Petherick et al, 2013) using the following antibodies: GPRC5A (1:2,000, CST, 12968), β‐actin (1:10,000, Sigma, A5316), HIF‐1α (1:1,000, BD, 610959), HIF‐1β (1:1,000, BD, 611078), HIF‐2α (1:1,000, CST, 7096), PLOD2 (1:1,000, R&D, MAB4445), CA9 (1:5,000, Novus, NB100‐417), cleaved PARP (1:20,000, Abcam, ab32064), active caspase‐3 (1:1,000, CST, 96645), p‐YAP S397 (1:5,000, CST, 13619), YAP (1:5,000, CST, 14074), BCL‐XL (1:1,000, BD, 556361), BCL‐2 (1:200, Santa Cruz, SC‐509), V5‐tag (1:2,000, CST, 13202), CYR61 (1:2,000, Santa Cruz, SC‐374129), RhoA (1:2,000, CST, 2117), lamin A/C (1:10,000, Sigma, 4C11) and α‐tubulin (1:10,000, Sigma, T6199). Cells were washed with ice‐cold PBS and lysed on ice for 10 min with Cell Signaling Technology lysis buffer (9803) supplemented with protease inhibitors and sonicated briefly. Equal protein concentrations were resolved using sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to an Immobilon‐P polyvinylidene difluoride membrane (Millipore). For GPRC5A Western blots, samples were not boiled.

Greenhough A., Bagley C., Heesom K.J., Gurevich D.B., Gay D., Bond M., Collard T.J., Paraskeva C., Martin P., Sansom O.J., Malik K, & Williams A.C. (2018). Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis. EMBO Molecular Medicine, 10(11), e8699.

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Western Blot Analysis of CYR61 in SMCs

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SMCs were lysed with radioimmunoassay precipitation buffer in the presence of protease inhibitors (Santa Cruz Biotechnology). Solubilized protein (30 μg) was loaded on a Mini-protean TGX gel (Bio-Rad) and underwent electrophoresis at 240V for 40 min using the Mini-PROTEAN Tetra Cell (Bio-Rad), transferred to a nitrocellulose membrane via a Bio-Rad Transblot Turbo, which was blocked with 5% milk buffer for 1 h at room temperature, rinsed with TBS plus 0.5% tween 20 and incubated with 1:750 of CYR61 (Santa Cruz Biotechnology) overnight in 5% milk buffer followed by 1:2500 of goat anti-mouse HRP (Santa Cruz Biotechnology) in 5% milk buffer the following day. Membranes were developed with Clarity ECL (Bio-Rad) and stained with Ponceau (Sigma) to visualize total protein. The membrane was scanned using Chemidoc™ XRS+system (Bio-Rad), and the bands were analyzed using ImageJ software [20 ]. SMCs from one participant were used per membrane with a total of three participants being assayed.

Wallace K., Chatman K., Johnson V., Brookins A., Rushing J, & LaMarca B. (2018). Novel treatment avenues for uterine leiomyoma: a new implication for endothelin?. Clinical science (London, England : 1979), 132(20), 2261-2267.

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Western Blot Analysis of Cellular Proteins

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Proteins were extracted from HO8910 and A2780 cells cultured for 30 min in a 37°C atmosphere containing 5% CO₂. The extracted proteins were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and electrophoretically transferred onto nitrocellulose membranes (Millipore, USA). Standard WB analyses were performed using antibodies specific for CTGF (Santa Cruz, CA, USA), CYR61 (Santa Cruz, USA), FN1 (Sigma), HMGA2 (Sigma), ASAP3 (Sigma, USA), ERBB3 (Sigma, USA), IL-6 (Sigma, USA), IL-1 (Sigma, USA), JUN (Invitrogen, USA), MAP2K8 (Sigma, USA), MMP13 (Sigma, USA), NPNT (Invitrogen, USA), ODC1 (Invitrogen, USA), VEGFC (Sigma, USA) and GAPDH (Santa Cruz, USA). Immunoreactions were visualized using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Santa Cruz, CA, USA) and detected by enhanced chemiluminescence. BandScan software was used to analyze the grayscale values.

ZHOU T., FU P., CHEN D, & LIU R. (2023). Transformer 2β regulates the alternative splicing of cell cycle regulatory genes to promote the malignant phenotype of ovarian cancer. Oncology Research, 31(5), 769-785.

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Protein Expression Analysis Using RIPA Lysis

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Cells were collected after washes with PBS, lysed in 100 μL of radioimmunoprecipitation (RIPA) buffer containing protease inhibitors (Cat# 04693116001, Roche, Mannheim, Germany) and sonicated. Protein concentrations were determined using the Pierce^TM BCA Protein Assay kit (Cat# 23227, Thermo Fisher Scientific, IL, USA). Primary antibodies against GAPDH (Cat# 2118, RRID: AB_561053, Cell Signaling Technology, MA, USA), YAP1 (Cat# 4912, RRID: AB_2218911, Cell Signaling Technology, MA, USA), p-YAP1 (S127) (Cat# 4911, RRID: AB_2218913, Cell Signaling Technology, MA, USA), CYR61 (Cat# sc-374129, RRID: AB_10947399, Santa Cruz Biotechnology, CA, USA), CCNA2 (Cat# NBP1-31330, RRID: AB_10003781, Novus Biologicals, CO, USA), E2F1 (Cat# A300-766A, RRID: AB_2096774, Bethyl Laboratories, TX, USA), and FOXM1 (Cat# A301-533A, RRID: AB_999586, Bethyl Laboratories, TX, USA) were used.

Baek S.W., Mun J.Y., Jang I.H., Yang G.E., Jeong M.S., Kim S.K., Nam J.K., Chu I.S, & Leem S.H. (2022). YAP1 activation is associated with the progression and response to immunotherapy of non-muscle invasive bladder cancer. eBioMedicine, 81, 104092.

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Verteporfin-Mediated Cell Signaling Pathways

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Verteporfin (Visudyne) was obtained from Novartis (Novartis, Basel, Switzerland) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was purchased from Sigma Aldrich (St.Louis, MO, USA). The following primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and were diluted 1:1000 unless stated otherwise: c-myc, axl, phospho-S6 ribosomal protein (Ser235/236), phospho-4EBP1 (Thr37/46), LC3B, phospho-p38 MAPK (Thr180/Tyr182), 4-Oct, survivin, pAkt (S473) (1:2000). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA): cyr61 (1:500), VEGFA (1:500), CTGF (1:500).

Al-Moujahed A., Brodowska K., Stryjewski T.P., Efstathiou N.E., Vasilikos I., Cichy J., Miller J.W., Gragoudas E, & Vavvas D.G. (2017). Verteporfin inhibits growth of human glioma in vitro without light activation. Scientific Reports, 7, 7602.

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Western Blot Analysis of Cell Signaling Proteins

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Proteins in whole cell lysates were separated with SDS-PAGE and then transferred to nitrocellulose membrane (Millipore). Primary antibodies including β-catenin (Beyotime, China, AF0069), MRTF-A (proteintech, 21166-1-AP), Cyr61(Santa Cruz, sc-374129), MYL9 (Santa Cruz, sc-28329) and GAPDH (Santa Cruz). Secondary antibodies were IRDye-conjugated donkey anti-mouse or anti-rabbit IgG (Licor Biosciences) and membranes were visualized with Odyssey Infrared Imaging System (Gene Company Limited).

He H., Du F., He Y., Wei Z., Meng C., Xu Y., Zhou H., Wang N., Luo X.G., Ma W, & Zhang T.C. (2018). The Wnt-β-catenin signaling regulated MRTF-A transcription to activate migration-related genes in human breast cancer cells. Oncotarget, 9(20), 15239-15251.

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Western Blot Analysis of Pancreatic Proteins

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Whole pancreata were lysed in RIPA buffer by TissueLyser LT (Qiagen, Hilden, Germany). Lysates were loaded to SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA). Blots were sequentially incubated with 5% BSA, primary antibody (CYR61: Santa Cruz Biotechnology; GAPDH: Sigma-Aldrich; E-cadherin and N-cadherin: BD Biosciences; Vimentin: DAKO), HRP-labeled secondary antibody (DAKO). Signals were revealed with Luminata Western HRP Substrate (Millipore, Schaffhausen, Switzerland) and detected with X-Ray films.

Huang Y.T., Lan Q., Ponsonnet L., Blanquet M., Christofori G., Zaric J, & Rüegg C. (2015). The matricellular protein CYR61 interferes with normal pancreatic islets architecture and promotes pancreatic neuroendocrine tumor progression. Oncotarget, 7(2), 1663-1674.

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