Phospho src

Manufactured by Santa Cruz Biotechnology

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Phospho-Src is an antibody that detects the phosphorylated form of the Src protein. Src is a non-receptor tyrosine kinase that plays a critical role in various cellular processes. The phosphorylation of Src is an important regulatory mechanism that modulates its activity and function.

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Anti-phospho-Src (2 citations)

6 protocols using phospho src

CXCL1 Signaling Pathway Activation

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Cells were seeded in 6 well plates at 250,000/well for 24 hours, serum starved for 24 hours, and stimulated with 60 ng/ml mouse recombinant CXCL1 (RnD Systems) for up to 24 hours. Cells were lysed in RIPA buffer containing protease inhibitors (Sigma cat no. P8340) and 10 μM sodium orthovanadate and sonicated. 50 μg protein were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were blocked in PBS containing 0.05% Tween-20 (PBS-T) containing 3% dry milk for 1 hour and immunoblotted with primary antibodies at a 1:1000 overnight in PBS-T/3% BSA. With the exception of FAK, antibodies were obtained from Cell Signaling Technology: phospho-Src (Tyr416 cat no. 6943), Src (cat no. 2123), phospho-FAK (Tyr397 cat no.8856), FAK (Santa Cruz Biotechnology, cat no. sc558), phospho-p38MAPK (Thr180/Tyr182, cat no. 4511), p38MAPK (cat no. 9212), phospho-IκB (Ser32/36, cat no. 9246), IκB (cat no. 2678), phospho-p42/44MAPK (Thr202/Tyr204, cat no. 9101), p42/44MAPK (cat no. 9102), phospho-AKT (Ser473, cat no. 4060), AKT (cat no. 2965), phospho-Stat3 (Tyr705, cat no. 9145), Stat3 (cat no. 9132). Immunoblots were washed in PBS-T 3 times for 10 minutes each and incubated with secondary anti-rabbit conjugated to horse radish peroxidase at a 1:1000 dilution in PBS-T/3% milk for 2 hours. Reactions were catalyzed with West Femto ECL substrate.

Bernard S., Myers M., Fang W.B., Zinda B., Smart C., Lambert D., Zou A., Fan F, & Cheng N. (2018). CXCL1 derived from mammary fibroblasts promotes progression of mammary lesions to invasive carcinoma through CXCR2 dependent mechanisms. Journal of mammary gland biology and neoplasia, 23(4), 249-267.

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Comprehensive Protein Expression Analysis

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Cells and resected tumor tissues were subjected to protein extraction using tissue protein reagents (T-PER, Pierce Biotechnology, Rockford, IL, USA). Proteins were separated by SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with the following antibodies: cyclin D1, β-catenin, Src, phospho-Src, Akt, phospho-Akt, Smad2/3, phospho-Smad2/3, Thromboxane A2 Receptor (TXA2R) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Glucose Transporter-1 (GLUT1), PKM2, SLC1A5, SLC7A5, glutaminase, CD41, CD62P, CD45, SDF-1α, CXCR4 (Abcam, Cambridge, MA, USA), and Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) (R&D Systems, Minneapolis, MN, USA). The reacted proteins were further determined with horseradish peroxidase-labeled IgG, visualized using Enhanced Chemiluminescence (ECL) Western blotting reagents, and quantified by the optical densitometry (Image Master ID, Pharmacia Biotech, Upsalla, Sweden) of developed autoradiographs.

Wang J.D., Chen W.Y., Li J.R., Lin S.Y., Wang Y.Y., Wu C.C., Liao S.L., Ko C.C, & Chen C.J. (2020). Aspirin Mitigated Tumor Growth in Obese Mice Involving Metabolic Inhibition. Cells, 9(3), 569.

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Immunoprecipitation and Western Blot Analysis

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Cell lysates were collected in radio-immunopreicipitation assay buffer and subjected to western blotting analysis (40 μg/lane) as described previously.^{36 (link),37 (link)} For immunoprecipitation, 500 μg of soluble protein was first incubated with primary antibodies for 2 h at room temperature and further incubated overnight at 4 °C after addition of 25 μl of protein A/G-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immunoprecipitated proteins were properly washed and separated by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) western blotting analysis. The following antibodies were used in this study: β-actin (Sigma-Aldrich, St Louis, MO, USA), ATG-5 (Santa Cruz Biotechnology), phospho-Src (Tyr416), Src, LC3, phospho-Akt (Ser473), Akt, phospho-mTOR (Ser2448), mTOR, phospho-S6 (Ser235/236), S6, phospho-AMPKα (Thr172), phosphor-Raptor (Serine 792), and AMPKα (Cell Signaling Technology, Beverly, MA, USA).

Nguyen H.G., Yang J.C., Kung H.J., Shi X.B., Tilki D., Lara PN J.r., DeVere White R.W., Gao A.C, & Evans C.P. (2014). Targeting autophagy overcomes Enzalutamide resistance in castration-resistant prostate cancer cells and improves therapeutic response in a xenograft model. Oncogene, 33(36), 4521-4530.

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Western Blot Analysis of Focal Adhesion Signaling

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Cells were lysed in radioimmunoprecipitation assay lysis buffer. Total proteins were separated via 10–15% sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes, blocked in 5% non-fat skim milk in TBST (10 mM tris, 150 mM NaCl, and 0.05% Tween 20), and subsequently incubated with the following primary antibodies: phospho-FAK, FAK (Cell Signaling Technology, Beverly, MA, USA), phospho-Src, Src, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Relative expression level of proteins was quantified using the Fusion Solo Detector (Vilber Lourmat, Marne-la-Vallée, France).

Lee S.G., Kim J.S., Lee H.S., Lim Y.M., So J.H., Hahn D., Ha Y.S, & Nam J.O. (2017). Bioconverted Orostachys japonicas Extracts Suppress Angiogenic Activity of Ms-1 Endothelial Cells. International Journal of Molecular Sciences, 18(12), 2615.

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Molecular Signaling Pathway Analysis

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The acarbose was purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, Penicillin-Streptomycin-Amphotericin B (PSA), fetal bovine serum (FBS), pyruvate and trypsin EDTA were purchased from Hyclone (Logan, UT, USA) and Gibco (Grand Island, NY, USA) for cell culture. The ammonium persulfate (APS), sodium dodecyl sulfate (SDS), bisacrylamide, TEMED, PVDF membranes, and NC membranes were purchased from Gibco (Grand Island, NY, USA) and Hyclone (Logan, UT, USA) for Western blot analysis. The antibodies to Ras, phospho-ERK, ERK, PCNA, PI3K, phospho-AKT, AKT, Rac1, RhoA, Cdc42, phospho-FAK, FAK, MMP9, MMP2, phospho-Src, Src and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Novus Biological (Littleton, CO, USA).

Chuang W.Y., Yu M.H., Yang T.Y., Chan K.C, & Wang C.J. (2020). Acarbose attenuates migration/proliferation via targeting microRNA-143 in vascular smooth muscle cells under diabetic conditions. Journal of Food and Drug Analysis, 28(3), 461-474.

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Western Blot Analysis of Cell Signaling

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Cells were lysed in ice-cold Laemmli sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes which were incubated with specific antibodies followed by horseradish peroxidase-labeled IgG. All antibodies were reacted with fibronectin, integrin α5, integrin β1, FAK, phospho-FAK (Tyr-397), ERK, phospho-ERK (Thr-202/Tyr-204), Akt, phospho-Akt (Ser-473), Src, phospho-Src (Tyr-416), cyclin D1, vimentin, Smad (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Smad (Ser-423/425, Cell Signaling, Danvers, MA, USA), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (R&D Systems, Minneapolis, MN, USA). The signals in membranes were visualized using enhanced chemiluminescence (ECL) Western blotting reagents and the intensity of signals was determined by a computer image analysis system (Alpha Innotech Corporation, IS1000, San Leandro, CA, USA).

Ou Y.C., Li J.R., Wang J.D., Chang C.Y., Wu C.C., Chen W.Y., Kuan Y.H., Liao S.L., Lu H.C, & Chen C.J. (2019). Fibronectin Promotes Cell Growth and Migration in Human Renal Cell Carcinoma Cells. International Journal of Molecular Sciences, 20(11), 2792.

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