C src

Manufactured by Cell Signaling Technology

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C-Src is a non-receptor tyrosine kinase that plays a crucial role in various cellular processes, including cell growth, differentiation, and survival. It is a key component of signal transduction pathways and is involved in the regulation of a wide range of cellular functions.

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Anti-phospho-c-Src (6 citations) Anti-c-Src (6 citations) PY416-c-Src (4 citations) Phospho-c-Src (4 citations) C-Src (p-c-Src) (2 citations) P‐c‐Src (2 citations)

20 protocols using c src

Signaling Pathway Analysis Protocol

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The Dulbecco’s modified Eagle medium (DMEM)/F-12 medium, fetal bovine serum (FBS), and TRIzol were from Invitrogen (Carlsbad, CA, USA). The hybond membrane and enhanced chemiluminescence (ECL) western blotting detection system were from GE Healthcare Biosciences (Buckinghamshire, UK). The anti-phospho antibodies against p42/p44 MAPK (#9101), p38 MAPK (#9211), JNK1/2 (#4668), mTOR (#5536), Akt (#9271), c-Src (#2101), Pyk2 (#3291), PKCα/βII (#9375), PKCδ (#9374), p65 (#3031), FoxO1 (#9461), and mTOR (#2972) were from Cell Signaling (Danvers, MA, USA). The antibodies against p44 MAPK (sc-94), p42 MAPK (sc-154), p38 MAPK (sc-535), JNK1/3 (sc-474), JNK2 (sc-827), Akt (sc-8312), c-Src (sc-18), PKCα (sc-208), PKCδ (sc-213), and p65 (sc-7151) were from Santa Cruz (Santa Cruz, CA, USA). The antibody against GAPDH (#MCA-1D4) was from EnCor (Gainesville, FL, USA). The antibodies against Pyk2 (ab55358) and FoxO1A (ab52857) were from Abcam (Cambridge, MA, USA). Galangin (3,5,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) was purchased from Cayman Chemicals (Ann Arbor, MI, USA). The thrombin, enzymes, and other chemicals were from Sigma (St. Louis, MO, USA). The SDS-PAGE reagents were from MDBio Inc (Taipei, Taiwan).

Yang C.C., Lin C.C., Hsiao L.D, & Yang C.M. (2018). Galangin Inhibits Thrombin-Induced MMP-9 Expression in SK-N-SH Cells via Protein Kinase-Dependent NF-κB Phosphorylation. International Journal of Molecular Sciences, 19(12), 4084.

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Immunoblotting Analysis of Cell Signaling

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Cells after SC99 treatment were prepared for immunoblotting according to our previous method [35 (link), 40 (link)]. A specific primary antibody against cyclin D₂ (CCND2) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); antibodies against Bcl-2, Bcl-xL, Caspase-3, STAT3, p-STAT3(Tyr705), c-Src, p-Src, JAK2, p-JAK2, E2F-1, AKT, p-AKT, ERK, p-ERK, mTOR, p-mTOR, and PARP were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against VEGF, β-actin, α-tubulin, anti–mouse immunoglobulin G (IgG) and anti–rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems (Minneapolis, MN). Anti-Myc antibody and GAPDH was purchased from Sigma (St. Louis, MO).

Zhang Z., Mao H., Du X., Zhu J., Xu Y., Wang S., Xu X., Ji P., Yu Y., Cao B., Han K., Hou T., Xu Z., Kong Y., Jiang G., Tang X., Qiao C, & Mao X. (2016). A novel small molecule agent displays potent anti-myeloma activity by inhibiting the JAK2-STAT3 signaling pathway. Oncotarget, 7(8), 9296-9308.

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Western Blotting and Immunoprecipitation Antibodies

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For Western blotting and immunoprecipitation, antibodies used were as
follows: 12-LOX (Oxford Biomedical Research, Rochester Hills, MI),
Phosphotyrosine-20 (BIOMOL, Plymouth, PA), Phosphorylated c-Src-Y418 (pSrc)
(Invitrogen), c-Src (Cell Signaling), Phospho-specific Integrin β4-Y1494
(ECM Biosciences, Versailles, KY), β4 integrin subunit (BD Pharmingen),
and actin (Chemicon, Billerica, MA). An antibody to integrin β4 (3E1)
for immunoprecipitation and integrin stimulation [41 (link)] was also obtained from Chemicon.

Dilly A.K., Tang K., Guo Y., Joshi S., Ekambaram P., Maddipati K.R., Cai Y., Tucker S.C, & Honn K.V. (2016). Convergence of Eicosanoid and Integrin Biology: Role of Src in 12-LOX activation. Experimental cell research, 351(1), 1-10.

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Osteoclast Differentiation Mechanism

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Foetal bovine serum and Minimum Essential Medium Eagle Alpha Modification (α‐MEM) were obtained from Gibco (Sydney, Australia). Dehydrocostus lactone was purchased from Selleck (Shanghai, China), and Cell Counting Kit‐8 (CCK‐8) was obtained from Dojindo Molecular Technologies (Shanghai, China). Recombinant murine RANKL and M‐CSF were purchased from R&D Systems (Minneapolis, MN). Primary antibodies against extracellular signal‐regulated kinase (ERK), p‐ERK, c‐Jun N‐terminal kinase (JNK), p‐JNK, p38, p‐p38, p65, p‐p65, IκBα, p‐IκBα, IκB kinase β (IKKβ), p‐IKKα/β, NFATc1/NFAT2, c‐Src, and β‐actin were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against cathepsin K were obtained from Abcam (Cambridge, MA). Rhodamine‐conjugated phalloidin was from Cytoskeleton (Denver, CO). Titanium particles were purchased from Alfa Aesar (#00681; Alfa Aesar, Ward Hill, MA). The TRAP staining kit, Tris, glycine, dimethylsulfoxide (DMSO), and sodium dodecyl sulfate were from Sigma‐Aldrich (St. Louis, MO) unless otherwise stated.

Hu B., Wu F., Shi Z., He B., Zhao X., Wu H, & Yan S. (2019). Dehydrocostus lactone attenuates osteoclastogenesis and osteoclast‐induced bone loss by modulating NF‐κB signalling pathway. Journal of Cellular and Molecular Medicine, 23(8), 5762-5770.

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Immunoprecipitation of Protein Complexes

Cited 1 time

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Protein extraction from both yeast and mammalian cells was carried out using methods previously described (Mollapour et al., 2010 (link)). For immunoprecipitation, mammalian cell lysates were incubated with anti-FLAG or anti-HA antibody conjugated agarose beads (Sigma) for 2 h at 4°C. Immunopellets were washed 4 times with fresh lysis buffer (20mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl₂, 0.1% NP40, protease inhibitor cocktail (Roche), and PhosSTOP (Roche)) and eluted in 5x Laemmli buffer. Precipitated proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Co-immunoprecipitated proteins were detected by immunoblotting with antibodies recognizing FLAG, 6x-His (ThermoFisher Scientific), Hsp90-835-16F1, GAPDH, p23 (ENZO Life Sciences), Tsc1, FLCN, GR, Myc, V5, GAPDH, Hsp90α, FNIP2, c-Src (Cell Signalling), phospho-tyrosine, v-Src (Millipore), FNIP1, FNIP2 (NCI), FNIP1 (antibodies-online.com), Aha1 (StressMarq Biosciences), HA (Roche). Secondary antibodies raised against mouse, rabbit, and rat (Cell Signaling) and goat (Santa Cruz Biotechnology) were used (See Key resources table).

Backe S.J., Sager R.A., Regan B.R., Sit J., Major L.A., Bratslavsky G., Woodford M.R., Bourboulia D, & Mollapour M. (2022). A specialized Hsp90 co-chaperone network regulates steroid hormone receptor response to ligand. Cell reports, 40(2), 111039.

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Integrin and Signaling Pathway Analysis

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Synthetic androgen R1881 was from Perkin-Elmer. Enzalutamide was from Selleck. The following rabbit Abs (pAbs) were used: AR (N20), phospho-ERK1 (Santa Cruz); Smad3 (Invitrogen); p-src (Y416), Akt, p-Akt, p-JNK and p-p38 (Cell Signaling); prostate specific antigen (PSA) (DAKO Cytomation). A goat Ab against p38 was from Santa Cruz. A rabbit monoclonal Ab against β₆ integrin, B1, was from Dr. Dean Sheppard. The following mouse monoclonal Abs (mAbs): AR (441) (Santa Cruz); β₁ integrin (C-18) and JNK (BD Biosciences); c-src (Cell Signaling); β₁ integrin (TS2/16) and β₃ integrin (AP3) are from ATCC; β₅ integrin (P1F6) from Life Technologies, Inc.; α_v integrin (VNR147) and α₅ integrin (P1D6); CK8 (Boehringer Mannheim), CK18 (Sigma); β₆ integrin Abs 6.2A1, ch2A1 and 6.3G9 were previously described (21 (link)); β₆ integrin Ab 10D5 from Chemicon. Purified non-immune mouse IgGs (mIgG) from Pierce, 1E6 (IgG1) from Biogen and 1C10 were used as negative controls.

Lu H., Wang T., Li J., Fedele C., Liu Q., Zhang J., Jiang Z., Jain D., Iozzo R.V., Violette S.M., Weinreb P.H., Davis R.J., Gioeli D., FitzGerald T.J., Altieri D.C, & Languino L.R. (2016). αvβ6 Integrin Promotes Castrate-Resistant Prostate Cancer Through JNK1-Mediated Activation of Androgen Receptor. Cancer research, 76(17), 5163-5174.

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Comprehensive Signaling Pathway Analysis

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Specific antibodies against PARP, Caspase 3, p-AKT(S473), AKT, p-mTOR(Ser2448), p-mTOR(Ser2481), mTOR, Raptor, Rictor, p-P70S6K, P70S6K, p-4E-BP1, 4E-BP1, ERK (p42/p44), p-ERK (p42/p44), p-p38, p-JNK, c-Src, and p-Src were purchased from Cell Signaling Technologies, Inc. GAPDH antibody was purchased from Abgent (Suzhou, China). Antibodies against β-actin and α-tubulin, as well as anti-mouse immunoglobulin G (IgG) and anti-rabbit IgG horseradish peroxidase conjugated antibody were purchased from R&D Systems.

Han K., Xu X., Xu Z., Chen G., Zeng Y., Zhang Z., Cao B., Kong Y., Tang X, & Mao X. (2015). SC06, a novel small molecule compound, displays preclinical activity against multiple myeloma by disrupting the mTOR signaling pathway. Scientific Reports, 5, 12809.

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Immunoblotting for Signaling Pathways

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SP600125 (SP) was purchased from LC Laboratories. Murine monoclonal (m) antibodies (Abs) against the following antigens were used: human β1, TS2/16 (ATCC); β1, clone-18; JNK1/JNK2 (BD Pharmingen); c-Src (Cell Signaling). Rabbit polyclonal Abs against the following antigens were used: IGF-IR (IGF-IR-β sc713); AKT; FAK; ERK1/2 (Santa Cruz); chromogranin (Invitrogen); FAK^pY397, Src^pY416, JNK^{pT183, pY185}, AKT^pS473 and AKT (Cell Signaling). Non-immune rabbit IgG was purchased from Pierce. Alexa Fluor 488 goat anti-rabbit IgG was purchased from Invitrogen.

Sayeed A., Lu H., Liu Q., II D.D., Duffy A., McCue P., Dicker A.P., Davis R.J., Gabrilovich D., Rodeck U., Altieri D.C, & Languino L.R. (2016). β1 integrin- and JNK-dependent tumor growth upon hypofractionated radiation. Oncotarget, 7(33), 52618-52630.

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Corilagin Modulates Osteoclast Formation

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Alpha‐modified minimal essential medium (α‐MEM) and foetal bovine serum (FBS) were purchased from Thermo Fisher Scientific (Scoresby). Corilagin (Figure 1A) with a purity greater than 98% was purchased from Tongtian Biotechnology Co. and dissolved in different concentrations of 0‐2 µmol/L with dimethyl sulphoxide (DMSO), respectively. Primary antibodies against ERK, JNK, p38, p65, IkBα, p‐ERK, p‐PI3K, TRAF6, p‐TAK1, p‐JNK, p‐p38, p‐p65, p‐IkBα, GAPDH, NFATc1/NFAT2, c‐Fos and c‐Src were purchased from Cell Signaling Technology, while second antibodies were purchased from Boster Biological Technology Co.. Recombinant RANKL and recombinant M‐CSF were obtained from Novoprotein Scientific Inc (Shanghai, China). Tartrate‐resistant acid phosphatase (TRAP) staining kit and all other reagents were purchased from Sigma‐Aldrich, unless stated otherwise.

Lu J., Ye C., Huang Y., Huang D., Tang L., Hou W., Kuang Z., Chen Y., Xiao S., Yishake M, & He R. (2020). Corilagin suppresses RANKL‐induced osteoclastogenesis and inhibits oestrogen deficiency‐induced bone loss via the NF‐κB and PI3K/AKT signalling pathways. Journal of Cellular and Molecular Medicine, 24(18), 10444-10457.

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Epithelial-Mesenchymal Transition Regulation

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TAM and 18α-GA were provided by Sigma-Aldrich (St. Louis, MO, USA). TGF-β1 was obtained from Pepro Tech (Rocky Hill, NJ, USA). MS-275 was supplied by MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against Cx43 and α-SMA were obtained from Abcam (Cambridge Science Park, Cambridge, England). E-ca, N-ca, p-Akt, Akt, c-Src and p-c-Src antibodies and LY294002 (PI3K inhibitor) were purchased from Cell Signaling Technology (Danvers, MA, USA). β-actin and GAPDH antibodies were purchased from Bioworld Technology, Co, Ltd. (Nanjing, China). Dasatinib was from Aladdin Bio-Chem Technology Co.LTD (Shanghai, China). IGF-1 was from PeproTech, Inc. (Rocky Hill, USA). Other reagents were all provided from Sigma-Aldrich unless otherwise noted.

Wu D.P., Zhou Y., Hou L.X., Zhu X.X., Yi W., Yang S.M., Lin T.Y., Huang J.L., Zhang B, & Yin X.X. (2021). Cx43 deficiency confers EMT-mediated tamoxifen resistance to breast cancer via c-Src/PI3K/Akt pathway. International Journal of Biological Sciences, 17(10), 2380-2398.

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