Anti phospho src tyr416

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-phospho-Src (Tyr416) is a primary antibody that specifically recognizes the phosphorylated form of Src kinase at tyrosine 416. This antibody is designed for use in Western blotting and immunohistochemistry applications.

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Lab products found in correlation

Anti src, by Cell Signaling Technology (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Anti src 36d10, by Cell Signaling Technology (1 mentions) Immuno blot pvdf membrane, by Bio-Rad (1 mentions) Anti actin, by Abcam (1 mentions) Western lightning plus ecl kit, by PerkinElmer (1 mentions) 4 amino 5 4 chlorophenyl 7 t butyl pyrazolo 3 4 d pyrimidine pp2, by Merck Group (1 mentions) Anti pkc, by BD (1 mentions) Streptavidin fitc, by Thermo Fisher Scientific (1 mentions) Alexafluor594 chicken anti rabbit, by Thermo Fisher Scientific (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Anti fgf2, by R&D Systems (1 mentions) Anti fgfr1, by Cell Signaling Technology (1 mentions) Anti mouse hrp, by Agilent Technologies (1 mentions) Anti rabbit hrp, by Agilent Technologies (1 mentions) Anti fgf 2, by Thermo Fisher Scientific (1 mentions) Anti β5, by Cell Signaling Technology (1 mentions) Dapi prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions) Anti pan cytokeratin, by Agilent Technologies (1 mentions) Anti gapdh, by Merck Group (1 mentions)

8 protocols using anti phospho src tyr416

Antibody-Based Techniques for Cellular Analysis

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The following antibodies against human were used for Western blotting and immunoprecipitation technic: Anti-GAPDH (Sigma), anti-phospho-SRC Tyr416 and anti-SRC (Cell Signaling), anti-FGF-2 (Thermo Fisher Scientific Inc.), anti-FGFR1 and anti-β₅ (Cell Signaling), all at 1/1000 dilution. Secondary antibodies used were: anti-rabbit-HRP and anti-mouse-HRP (Dako), both at 1/1000 dilution.
For FACS analysis, anti-α_v-PE and anti-β₅ (Biolegend), anti-β₃-PE and anti-α_vβ₃-PE (BD Biosciences), anti-β₆ (clone IC8C3, Dr. D. Sheppard, UCSF, San Francisco) were used at 1/100 dilution. Anti-α_vβ₅ (clone P5H9, R&D Systems) and anti-α_vβ₆ (clone 10D5, Merck Millipore) were used for FACS at 1/50 dilution, and for function blocking experiments at 10 μg/ml, as well as anti-FGF-2 (R&D Systems) and anti-β₁ (clone Lia1/2, Beckman). Dead Cell Apoptosis Kit for PI-Annexin V staining was from Life technologies.
anti-phospho-SRC Tyr416 (Cell Signaling), anti-FGFR1 (Cell Signaling), anti-Phalloïdin-AlexaFluor 546 (Invitrogen), anti-FGF-2 (Thermo Fisher Scientific Inc.), as well as anti-αSMA (Sigma), anti-pan-Cytokeratin (Dako) and DAPI ProLong Gold mounting medium (Invitrogen) were used for immunofluorescent staining and immunohistochemistry.

Knuchel S., Anderle P., Werfelli P., Diamantis E, & Rüegg C. (2015). Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion. Oncotarget, 6(16), 14300-14317.

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Immunoblot Analysis of Phospho-Src

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Cell lysates (20 μg) were separated on 12.5% SDS–PAGE gels, and transferred to Immuno-blot PVDF membranes (BioRad). After blocking with 5% skim milk in a TBS-Tween 0.1% solution, the membrane was reacted with anti-phospho-Src (Tyr416; dilution 1:1000) and anti-Src (36D10; dilution 1:1000) antibodies (all from Cell Signaling Technology) at room temperature for 1 h. Anti-actin (Abcam) was used as a loading control. Membranes were subsequently incubated with goat anti-mouse or anti-rabbit HRP-conjugated IgG (Jackson ImmunoResearch laboratories) for 1 h and bound HRP was detected using a Western lightning plus-ECL kit (Perkin Elmer).

Oyama R., Takahashi M., Yoshida A., Sakumoto M., Takai Y., Kito F., Shiozawa K., Qiao Z., Arai Y., Shibata T., Araki Y., Endo M., Kawai A, & Kondo T. (2017). Generation of novel patient-derived CIC-DUX4 sarcoma xenografts and cell lines. Scientific Reports, 7, 4712.

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Peptide-Based aPKC Inhibition Assay

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aPKC pseudosubstrate peptide (Myr-SIYRRGARRWRKL(biotin)YCAN), scrambled aPKC (Myr-WRICGNKARL(biotin)RRYYSAR, and PKCα/β (Myr-RFARKGAL(biotin)RQKNVHEVKN) were synthesized by GenScript Corp. (Piscataway, NJ). aPKCι cDNA was generously provided by Dr. Trevor Biden (Garvan Institute of Medical Research, Sydney, Australia). The mouse embryo fibroblast cell line SYF was purchased from American Type Culture Collection (Manassas, VA). Primary and secondary antibodies were purchased from the indicated suppliers; anti-PKC from BD Biosciences (San Jose, CA); anti-Src and anti-phospho-Src (Tyr416) from Cell Signaling Technology, Inc. (Danvers, MA); Alexa Fluor 488 chicken antimouse antibody, Alexa Fluor 594 chicken anti-rabbit, and Hoechst 33342 from Life Technologies (Grand Island, NY). FITC-streptavidin was purchased from ThermoScientific (Pittsburgh, PA). 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine (PP2) was purchased from EMD Millipore (Billerica, MA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Tisdale E.J., Shisheva A, & Artalejo C.R. (2014). Overexpression of Atypical Protein Kinase C in HeLa Cells Facilitates Macropinocytosis via Src Activation. Cellular signalling, 26(6), 1235-1242.

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Sanguinarine: Platelet Activation Assay

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Sanguinarine (13-methyl(1,3) benzodioxolo (5,6-c) -1,3-dioxolo(4,5-i) phenanthridinium), (HPLC ≥ 98%, SA, Figure 1) (Absin, Shanghai, China), was dissolved in 0.1% DMSO (final concentration). The EDTA, prostaglandin E1(PGE1) and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). The CHRONO-LUME reagent and collagen were acquired from Chrono-Log Corp. (Havertown, PA, USA). The collagen-related peptide was obtained from Dr. Newman’s lab (Blood Center of Wisconsin, Milwaukee, WI, USA). The FITC-conjugated anti-CD62P (P-selectin) antibody and FITC-conjugated anti-PAC-1(αIIbβ3) antibody were purchased from Biolegend (San Diego, CA, USA). The Fluo-3AM was purchased from MCE (Shanghai, China). The CellTrace Calcein Green was purchased from Invitrogen (Carlsbad, CA, USA). The anti-phospho-PI3K(Ser1070), anti-PI3K, anti-phospho-Akt(Ser473), anti-phospho-GSK3β (Ser9), anti-phoshpo-PLCγ2 (Tyr1217), anti-β3, anti-phospho-β3 (Tyr474), anti-Src, anti-phospho-Src (Tyr416) and anti-phospho-Syk (Tyr525/526) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The anti-Syk, anti-PLCγ2, anti-Akt and antil-GSK3β were acquired from Santa Cruz Biotechnology (Dallas, TX, USA).

Shu D., Zhu Y., Lu M., He A.D., Chen J.B., Ye D.S., Liu Y., Zeng X.B., Ma R, & Ming Z.Y. (2021). Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation. Biomedicines, 9(5), 444.

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Immunoblotting for Phospho-Src Activation

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Protein was extracted by RIPA buffer containing protease and phosphatase inhibitors. Protein samples were equally loaded with 25 μg protein on 10% gels and separated at 120V. PVDF membranes with transferred proteins were blocked with 5% milk in TBST and immunoblotted with the following antibodies: Rabbit monoclonal anti-Phospho-Src (Tyr416) (#6943, Cell Signaling Technology, 1:1000, USA), Mouse monoclonal anti-ACTB (AC026, ABclonal, 1:100000, CN). Chemiluminescent was detected by Amersham Imager 600 (GE Life Sciences, USA).

Li X., Zhang C., Deng M., Jiang Y., He Z, & Qian H. (2023). EFNB1 levels determine distinct drug response patterns guiding precision therapy for B-cell neoplasms. iScience, 27(1), 108667.

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Immunoblotting for Cellular Signaling Proteins

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Cell pellets were lysed in RIPA buffer (50 mM Tris pH7.4, 140 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate) containing phosphatase and protease inhibitor tablets (Roche). Cell pellets were lysed for 20 minutes on ice, after which nuclear and cellular debris was removed by centrifugation at 13,000 RPM for 10 minutes at 4°C. 10–20 μg of protein lysates were electrophoresed on NuPage precast gels (Invitrogen) and immunoblotted with anti-NF1 (Bethyl Antibodies), anti-phospho-SRC (Tyr416) (Cell Signalling Technology [CST]), anti-SRC (CST), phospho-ERK (CST), phospho-MEK (CST) total ERK (Cell signaling Technology), total MEK (CST), phospho-HER2 (CST), total HER2 (Santa Cruz), phospho-Insulin Receptor/IGF1R (CST), total insulin receptor (CST), phospho-FAK (Tyr925) (CST), total FAK (CST). As loading controls, vinculin (Santa Cruz) and beta-actin (Sigma) were used. Membranes were then incubated in anti-IgG-HRP (CST) and chemiluminescent detection (Luminata Cresendo, Millipore).

McGivern N., El-Helali A., Mullan P., McNeish I.A., Paul Harkin D., Kennedy R.D, & McCabe N. (2017). Activation of MAPK signalling results in resistance to saracatinib (AZD0530) in ovarian cancer. Oncotarget, 9(4), 4722-4736.

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Protein Extraction and Quantification for Western Blot

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For cell sample preparation, cells were lysed with an NP-40 buffer with protease inhibitor cocktail (Roche, 11697498001). For tissue sample preparation, single-cell suspensions derived from mouse tumors were incubated with anti-CD11b antibody–conjugated microbeads (1:100; Miltenyi Biotech, 130-049-601) for 15 min at 4°C and separated by MACS column with a separator. The eluted cells were lysed with NP-40 buffer with protease inhibitor cocktail and phosphatase inhibitor (Thermo Fisher Scientific, 78428). Total protein (20 μg) was resolved by 4 to 15% precast SDS–polyacrylamide gel electrophoresis (Bio-Rad) and followed by transfer. Polyvinylidene difluoride membranes were blotted with anti–phosphoSrc (Tyr⁴¹⁶; Cell Signaling Technology, 6943), anti-Hck (1:1000; Cell Signaling Technology, 14643; ABclonal, A14537), anti-Lyn (1:1000; Cell Signaling Technology, 2796), anti-FLAG (1:1000; GenScript, A00187-100), anti–arginase 1 (1:500; Santa Cruz Biotechnology, sc-20150), or anti–glyceraldehyde-3-phosphate dehydrogenase (1:3000; Cell Signaling Technology, 5174) antibody overnight at 4°C. Proteins were detected with horseradish peroxidase–conjugated secondary antibodies (Bio-Rad) and enhanced by enhanced chemiluminescence development (GE Healthcare, RPN2232).

Yu X.C, & Margolin W. (2000). Deletion of the min Operon Results in Increased Thermosensitivity of an ftsZ84 Mutant and Abnormal FtsZ Ring Assembly, Placement, and Disassembly. Journal of Bacteriology, 182(21), 6203-6213.

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Immunofluorescence staining and RhoA biosensors

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The following antibodies were used for immunofluorescence staining: anti-E-cadherin (1:250; BD Biosciences), anti-β-catenin (1:250; BD Biosciences), anti-α-catenin (1:250; Sigma Aldrich), anti-Phospho-Src Tyr416 (1:50; Cell Signaling), Cy5 conjugated donkey anti-rabbit (1:800; Jackson Laboratory), and Cy3 conjugated goat anti-mouse (1:1000; Invitrogen). In addition, Alexa488 conjugated phalloidin (1:40; Invitrogen) was used to stain actin filaments. pRBD-YPet was constructed by inserting the cDNA sequence encoding aa 7–89 of mouse rhotekin into the EcoRI/BamHI sites of pYPet-N1. The YPet sequence was amplified from pCEP4YPet-MAMM (Addgene plasmid 14032) and cloned into AgeI/NotI sites of pEYFP-N1 (Clontech), generating the pYPet-N1 (Nguyen and Daugherty 2005 (link)). The pECFP-RhoA construct was previously described (Picard et al. 2009 (link)).

Gutierrez N., Eromobor I., Petrie R.J., Vedula P., Cruz L, & Rodriguez A.J. (2014). The β-actin mRNA zipcode regulates epithelial adherens junction assembly but not maintenance. RNA, 20(5), 689-701.

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