Caveolin 1

Manufactured by Cell Signaling Technology

Sourced in United States

Caveolin-1 is a protein that plays a key role in the formation and function of caveolae, specialized invaginations of the cell membrane. It is involved in the regulation of various cellular processes, including signal transduction, endocytosis, and cholesterol homeostasis.

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Lab products found in correlation

Close spelling variants of this product

Rabbit monoclonal anti-phosphorylated Caveolin-1 Anti-Caveolin-1 antibody Caveolin-1 antibody Anti-caveolin-1 Rabbit monoclonal anti- Caveolin-1 Anti-phospho-caveolin-1 (Tyr14) Rabbit anti-caveolin-1

52 protocols using caveolin 1

Western Blot Analysis of Cell Junctions

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Western blot analysis was performed as described previously29 (link). Briefly, protein concentrations were estimated by the Bradford method, equal amount of protein (100 μg/lane) was diluted in 10 × sample buffer, boiled, and loaded onto 12% SDS-PAGE gels and transferred to a nitrocellulose membrane (Hybond-C, Amersham Biosciences, USA). The membranes were incubated overnight at 4 °C with the primary antibodies against β-actin, occludin, ZO-1, MLC, MLCK, MLC (S20) (Abcam, Cambridge, UK), claudin-5, MMP-2, MMP-9 (Santa Cruz Biotechnology, Santa Cruz, USA), caveolin-1, Src, caveolin-1 and Src (Tyr416) (Cell Signaling, Beverly, Massachusetts, USA). Then the membranes were washed with TBST and incubated with the respective horseradish peroxidase-conjugated secondary antibodies at a 1:5000 dilution for 60 min at room temperature. The protein bands were detected by enhanced chemiluminescence, and the band intensities were quantified by densitometry and expressed as mean area density using the image J (Bethesda, MD, USA) software.

Mao X.W., Pan C.S., Huang P., Liu Y.Y., Wang C.S., Yan L., Hu B.H., Chang X., He K., Mu H.N., Li Q., Sun K., Fan J.Y, & Han J.Y. (2015). Levo-tetrahydropalmatine attenuates mouse blood-brain barrier injury induced by focal cerebral ischemia and reperfusion: Involvement of Src kinase. Scientific Reports, 5, 11155.

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Molecular markers for cell analysis

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Monoclonal antibodies to CD44, ZEB1, Caveolin-1, and GAPDH were obtained from Cell Signaling Technology (San Diego, CA, USA). CD44 construct was obtained from Asia-Vector Biotechnology (Shanghai, China). Secondary antibodies conjugated with HRP were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). CD44, ZEB1, Caveolin-1 small interfering RNAs (siRNAs) were synthesized by RioBio Co. (Guangzhou, China). The target sequences of siRNA are indicated in Table S1.

Zhou J., Du Y., Lu Y., Luan B., Xu C., Yu Y, & Zhao H. (2019). CD44 Expression Predicts Prognosis of Ovarian Cancer Patients Through Promoting Epithelial-Mesenchymal Transition (EMT) by Regulating Snail, ZEB1, and Caveolin-1. Frontiers in Oncology, 9, 802.

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Inflammatory Signaling Pathway Protocols

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Fetal bovine serum was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Rabbit anti-mouse MyD88, caveolin-1, and eNOS antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies were purchased from OriGene (Beijing, China). All primers were synthesized at Tsingke (Hangzhou, China). Ethylenediaminetetraacetic acid and phosphate-buffered saline (PBS) were purchased from Huaan Biotechnology Co., Ltd. (Hangzhou, China). Enzyme-linked immunosorbent assay (ELISA) kits for detecting mouse IL-6, IL-1β, IP-1, and TNF-α were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Luminex liquid chip was purchased from EMD Millipore (Billerica, MA, USA).

Xu L.Y., Cai W.R., Ma C.F., Shou Q.Y., Qian J.L, & Huseyin T.S. (2018). Qi-Dong-Huo-Xue-Yin Inhibits Inflammation in Acute Lung Injury in Mice via Toll-Like Receptor 4/Caveolin-1 Signaling. Evidence-based Complementary and Alternative Medicine : eCAM, 2018, 2373609.

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Immunofluorescence Analysis of Arterial Proteins

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Artery samples were fixed in 4% paraformaldehyde solution for paraffin embedding. Frozen tissues were embedded in the O.C.T. compound (Tissue-Tek). Sections were blocked in 5% goat serum for 1 h at room temperature, then incubated with primary antibody at 4 °C overnight. The primary antibodies used in this study was as follows. CD31 (1:100, #ab28364, Abcam; 1:100, #ab9498, Abcam), caveolin-1 (1:50, #3267, Cell Signaling Technology), Sp1 (1:50, #sc−420, Santa Cruz Biotechnology Inc.), Sp3 (1:50, #sc-28305, Santa Cruz Biotechnology Inc.), p-AMPKα (Thr172, 1:100, #50071, Cell Signaling Technology). After washing with phosphate buffered saline, samples were incubated with Alexa Fluor-labeled secondary antibody at room temperature for 1 h including Alexa Fluor 488 (1:200, #ab150077, Abcam; 1:200, #ab150113, Abcam), Alexa Fluor 594 (1:200, #ab150080, Abcam; 1:200, #ab150116, Abcam). Finally, nuclei were stained with 4,6’-diamidino-2-phenylindole (DAPI, #ab104139, Abcam) for 5 min at room temperature, and immunofluorescence was analyzed under a florescent microscope.

Lu H., Jiang X., He L., Ji X., Li X., Liu S., Sun Y., Qin X., Xiong X., Philipsen S., Xi B., Zhang M., Yang J., Zhang C., Zhang Y, & Zhang W. (2023). Endothelial Sp1/Sp3 are essential to the effect of captopril on blood pressure in male mice. Nature Communications, 14, 5891.

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Antibody Validation for AMPK Signaling

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Antibodies to caveolin-1, phospho-AMPKα T172, AMPKα, Histone H3, eNOS, and phospho-eNOS Ser1177 were from Cell Signaling Technology (Danvers, MA). Antibodies to LKB1 and GAPDH were purchased from Santa Cruz Biotechnology (Santa. Cruz, CA). Anti-HuR was purchased from Millipore (Temecula, CA). Anti-CD31 was from Abcam (Cambridge, UK). FuGene HD transfection reagent was from Roche Applied Science (Indianapolis, IN).

Zhang W., Wang Q., Wu Y., Moriasi C., Liu Z., Dai X., Wang Q., Liu W., Yuan Z.Y, & Zou M.H. (2014). Endothelial Cell-Specific LKB1 Deletion Causes Endothelial Dysfunction and Hypertension in Mice in vivo. Circulation, 129(13), 1428-1439.

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Detection of Flotillin, H2AX, and Caveolin

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Antibodies to detect flotillin-1γ, H2AX, and caveolin-1 were obtained from Cell Signaling Technologies (Danvers, MA) and antibodies to detect flotillin-2 and DHHC5 were obtained from Millipore Sigma (Oakville, ON). Dynasore and desipramine were obtained from Millipore Sigma. Lysine-fixable Alexa-488-conjugated dextran (A488-dextran, 10000 MW), and Alexa-488- phalloidin (A488-phalloidin) were obtained from Thermo Fisher Scientific (Waltham, MA). Latrunculin A was obtained from Cayman Chemical (Ann Arbor, MI).

Fekri F., Abousawan J., Bautista S., Orofiamma L., Dayam R.M., Antonescu C.N, & Karshafian R. (2019). Targeted enhancement of flotillin-dependent endocytosis augments cellular uptake and impact of cytotoxic drugs. Scientific Reports, 9, 17768.

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Monoclonal Antibody 9.2.27 in Cell Signaling

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The anti-CSPG4 monoclonal antibody 9.2.27 utilized for western blot, immunoprecipitation and flow cytometry in these studies was provided by Dr. Ralph Reisfeld (The Scripps Research Institute, La Jolla, CA). Other antibodies used for IP and western blot were purchased from the indicated companies: Tubulin specific antibody from Oncogene Research Products (Pasadena, CA); anti-β1 integrin, phosphorylated Erk 1,2 (pErk 1,2), total Erk 1,2, Caveolin-1, phosphorylated-Src (pSrc)(pY416), pSrc(pY526), phosphorylated FAK(pY397) (pFAK) and FAK specific antibodies from Cell Signaling Technology, Inc (Boston, MA). Anti-Src and syntenin specific antibodies were from Santa Cruz Biotechnology; normal mouse monoclonal IgG_2a, normal rabbit IgG and goat anti-mouse FC from ICN Pharmaceuticals (Aurora, OH); peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies from Jackson ImmunoResearch Laboratories (West Grove, PA). Methyl-β-cyclodextran was purchased from Santa Cruz Biotechnology. Human plasma fibronectin was purified as previously described [25 (link)].

Yang J., Price M.A., Wanshura L.E., He J., Yi M., Welch D.R., Li G., Conner S., Sachs J., Turley E.A, & McCarthy J.B. (2019). CSPG4 Enhanced Melanoma Motility and Growth Requires a Cysteine in the Core Protein Transmembrane Domain. Melanoma research, 29(4), 365-375.

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Lipid Raft Isolation from Adipocytes and Macrophages

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LR and non-LR fractions from adipocytes and macrophages were obtained by Optiprep gradient centrifugation using a detergent-free protocol, as described previously (46 (link)). Briefly, cell pellets were homogenized in buffer (250 mmol/L sucrose, 1 mmol/L EDTA, 500 mmol/L sodium bicarbonate, pH 11). After centrifugation (1000g, 10 minutes), the postnuclear supernatant fraction was added to 60% Optiprep to a final concentration of 35% Optiprep, overlaid on a discontinuous gradient of 5%–35% Optiprep, and centrifuged at 326,512g in a Beckman NVT 65.2 rotor for 90 minutes at 4°C. Nine 0.5 mL fractions were collected and subjected to immunoblotting using antibodies against TLR4 (Cell Signaling Technology, 1:1000, 14358), NOX2 (Invitrogen, Thermo Fisher Scientific, 1:1000, PA5-76034), and caveolin-1 (Cell Signaling Technology, 1:1000, 3238).

Han C.Y., Kang I., Omer M., Wang S., Wietecha T., Wight T.N, & Chait A. (2020). Serum amyloid A–containing HDL binds adipocyte-derived versican and macrophage-derived biglycan, reducing its antiinflammatory properties. JCI Insight, 5(20), e142635.

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Western Blot Analysis of Insulin Signaling

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Typically, 10 µg of protein was resolved by SDS–PAGE, transferred to PVDF membranes and immunoblotted as previously described [5 (link)]. Antibodies detecting TRARG1 (sc-292062 or sc-377025) and 14-3-3 (sc-1657) were from Santa Cruz Biotechnology. Antibodies against pHSL S660 (#4126), pp90RSK (#9344), p4EBP1 S65 (#9456), pTBC1D4 T642 (#4288S), pAKT S473 (#4051), pAKT T308 (#9275L), AKT (#4685), HA (#C29F4), GSK3α (#9338S), GSK3β (#9832S), pGSK3 Ser 9/21 (#8566S), pGS S641 (#3891) and Caveolin1 (#3267) were purchased from Cell Signaling Technology. Anti-α-tubulin (#T9026) was from Sigma–Aldrich. Antibody against TBC1D4 was produced as previously described [4 (link)].

Duan X., Norris D.M., Humphrey S.J., Yang P., Cooke K.C., Bultitude W.P., Parker B.L., Conway O.J., Burchfield J.G., Krycer J.R., Brodsky F.M., James D.E, & Fazakerley D.J. (2022). Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate. Biochemical Journal, 479(11), 1237-1256.

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Caveolin-1 Enrichment and Western Blotting

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After cells were incubated with the inhibitors, respectively, for 12 h, Caveolin-1 enriched membrane fractions were isolated as described above and the final samples were detected by western blotting. The protein samples were separated by SDS-PAGE gel and then electrotransferred to PVDF membranes. Subsequently, blots were subjected to immunostaining with antibodies against Caveolin-1 (Cell Signaling Technology, 1 : 800), Cavin-1 (ANBO, 1 : 500), and lectin-like oxLDL receptor (Lox-1, WuXi AppTec, 1 : 1000). After incubation for 1 h with a peroxidase-conjugated secondary antibody (Abbkine, 1 : 10000), bands were visualized by an ECL western blotting detection system (NDR, Israel). The band intensities were quantified using ImageJ software.

Li W., Yang X., Xing S., Bian F., Yao W., Bai X., Zheng T., Wu G, & Jin S. (2014). Endogenous Ceramide Contributes to the Transcytosis of oxLDL across Endothelial Cells and Promotes Its Subendothelial Retention in Vascular Wall. Oxidative Medicine and Cellular Longevity, 2014, 823071.

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