The antibodies used were phospho-Smad2 (Cell Signaling Technology, 138D4), Smad2/3 (BD, 610842), SMA (Sigma, A2547), MLC2 (Cell Signaling Technology, 3674), SRF (sc-335), phospho(T18/S19)-MLC2 (Cell Signaling Technology, 3674S), MRTF-A (sc-21558), MRTF-B (sc-47282), pan–ERK (BD, 610124), YAP (for immunofluorescence, clone 63.7, sc-101199; for ChIP, ab52771), phospho-Ser127-YAP (Cell Signaling Technology, 4911S), TEAD1/TEF-1 (BD, 610922), TEAD4/TEF3 (sc-101184), Actin (Cytoskeleton, AAN01), pan 14-3-3 (clone H-8, sc-1657), histone H3 (Abcam, ab1791); RNA Pol II CTD S5P (Covance, H14), Integrin β3 (ab119993), Integrin αV (ab179475), phospho-(Tyr416) Src (Cell Signaling Technology, 2101), Src (2109S), HA high affinity (3F10) (Roche, 11867431001), HA-peroxidase high affinity (3F10) (Roche, 12013819001), Flag-M2-peroxidase (Sigma, A-85292), and Flag (for ChIP, Sigma, F7425; for immunofluorescence, clone M2, F1804). Immunoblotting and immunofluorescence were by standard techniques. F-Actin was visualized with Texas Red-X phalloidin (Invitrogen), and nuclei were visualized with DAPI.
Phospho src tyr416
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Src (Tyr416) is an antibody that detects endogenous levels of Src only when phosphorylated at tyrosine 416.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt ser473, by Cell Signaling Technology (8 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (3 mentions)
Phospho akt thr308, by Cell Signaling Technology (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Vimentin, by Cell Signaling Technology (2 mentions)
Phospho src tyr 527, by Cell Signaling Technology (2 mentions)
Integrin β3, by Cell Signaling Technology (1 mentions)
Integrin αv, by Cell Signaling Technology (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Texas red x phalloidin, by Thermo Fisher Scientific (1 mentions)
Histone h3, by Abcam (1 mentions)
Pan erk, by BD (1 mentions)
Smad2 3, by BD (1 mentions)
Phospho smad2, by Cell Signaling Technology (1 mentions)
Control sirna, by Santa Cruz Biotechnology (1 mentions)
3 methyladenine 3 ma, by Merck Group (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Antibodies against beclin 1, by Cell Signaling Technology (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
17 protocols using phospho src tyr416
1
Immunoblotting and Immunofluorescence Analysis
2
Regulation of STAT3-Dependent Autophagy
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STAT3-dependent luciferase reporter plasmid was provided by Dr Ming Shi from our institute. Human HO-1, STAT3, ATG5 and Src siRNA and their control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Invitrogen-Life Technology (Beijing, China) and Ruibo Biotechnology (Guangzhou, China), respectively. The antibodies against Beclin-1, LC3B, phospho-Tyr416-Src, Src, phospho-Tyr705-STAT3 and STAT3 were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies against HO-1 and β-actin were obtained from Santa Cruz Biotechnology. The anti-ATG5 antibody, DOX, 3-Methyladenine (3-MA) and chloroquine were purchased from Sigma (St. Louis, MO, USA).
3
Signaling Pathway Antibody Protocol
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Primary antibodies against phospho-Tyr 397 FAK, total FAK, phospho-Ser 473 Akt, total Akt, phospho-Tyr 416 Src, total Src, vimentin, snail, slug, and ZEB1 were purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against anti-human S100P and claudin-3 (Abcam, San Francisco, CA, USA), SM-actin (Sigma-Aldrich, St. Louis, MO), Fibronectin, N-cadherin and E-cadherin (BD Biosciences, Bedford, MA) were obtained from their respective companies. Primary antibodies against GAPDH, FAK, and AKT inhibitors were purchased from Millipore (Billerica, MA). S100P ELISA kit was obtained from CircuLex (Woburn, MA).
4
Immunoblot Analysis of Cell Signaling
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Whole cell protein lysates were extracted using cell lysis buffer (Cell Signaling Technology) following the manufacturer’s instructions and protein concentration was determined using the Bradford Assay Kit (Sigma-Aldrich). Immunoblotting was performed according to standard methods. The following primary antibodies were used according to the manufacturer’s instructions: cortactin (#3502), phospho-cortactin Tyr421 (#4569), ERK1/2 (#4695), phoshpho-ERK1/2 Thr202/Tyr204 (#4370), GAPDH clone D16H11 (#5174), Src (#2109) and phospho-Src Tyr416 (#2101), gelsolin D9W8Y (#12953) (all rabbit host species and obtained from Cell Signaling Technology) and E-cadherin NCH-38 (#MA5-12547, mouse, Thermo Fisher Scientific). After Secondary HRP-labeled antibody (rabbit #1706515, mouse #1706516, both obtained from Bio-Rad Laboratories) incubation (1 h, RT) immunolabeling was detected using an enhanced Chemiluminescence Detection Kit (SignalFire ECL Reagent; Cell Signaling Technology) and the Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad Laboratories).
5
Murine TRAMP and Human PC3 Cell Lines
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Murine TRAMP (TR-C2N) cell line (gifted by Dr. Warren Heston, Cleveland Clinic), human PC3 cells (ATCC, Manassas, VA) and “SYF” cells (mouse embryonic fibroblasts deficient in Src, Yes and Fyn) were maintained in DMEM (HyClone, Thermo Scientific, Logan, UT) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin in a 5% CO2 atmosphere at 37°C. Primary antibodies against phospho-AktSer473, phospho-GSK-3α/βSer9/21, phospho-SrcTyr416, phospho-βcateninSer33/37/Thr41, pan-Akt and siRNA for GSK-3α/β were purchased from Cell Signaling (Boston, MA). Phospho-GSK-3Tyr216 was obtained from BD Biosciences (Franklin Lakes, NJ). Primary antibodies against β-actin and GSK-3 inhibitor (SB415286) were purchased from Sigma, St. Louis, MO. All secondary antibodies were obtained from Bio-Rad (Hercules, CA). EGF was obtained from R&D (Minneapolis, MN). Docetaxel was purchased from Sigma, St. Louis, MO and Dasatinib was purchased from Santacruz biotechnology, (Dallas, TX).
6
Insulin Signaling Pathway Analysis
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α-minimal essential medium, fetal bovine serum and antibiotic/antimycotic solution were from Life Technologies. All other reagent-grade chemicals, including BSA and fatty acids, were obtained from Sigma–Aldrich unless otherwise stated. Wortmannin and okadaic acid were from Calbiochem-Merck. Fraction V fatty acid-free BSA was from Roche. Insulin was from R&D systems. Complete protein phosphatase inhibitor tablets were purchased from Roche Diagnostics. Phospho-AktThr308, phospho-AktSer473, phospho-ERK1/2Thr202/Tyr204, phospho-GSK3Ser21/9, phospho-CREBSer133, phospho-SrcTyr416, anti-Akt and anti-ERK1/2 antibodies were from Cell Signaling Technology. Anti-GSK3 and anti-PTEN antibodies were from Santa Cruz Biotechnology. Anti-IR (insulin receptor), anti-p85, anti-IRS1 and anti-PP2A subunit C, demethylated were from Millipore. Anti-β actin and anti-GAPDH were from Sigma. Phospho-PP2AY307 was from Abcam.
7
Protein Expression Analysis of Tumor Samples
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Total protein lysates from tumors were extracted by RIPA lysis buffer supplemented with proteinase and phosphatase inhibitors (Roche Diagnostics, 4693159001). The samples were separated on a 10% SDS‒PAGE gel, transferred to a nitrocellulose membrane (Millipore), and probed with the following antibodies: Fgfr2 (1:1000, Merck, sc-6930), phospho-Fgfr (1:1000, Cell Signaling Technology, 3476), Vegfr2 (1:1000, Cell Signaling Technology, 12599), phospho-Src (Tyr416) (1:1000, Cell Signaling Technology, 2101), β-actin (1:1000, Cell Signaling Technology, 3700), phospho-AKT (Ser473) (1:1000, Cell Signaling Technology, 9271), phospho-AKT (Thr308) (1:1000, Cell Signaling Technology, 13038), and EGFR (1:1000, Cell Signaling Technology, 8339). Full and uncropped western blots are presented in the Supplemental File.
8
Immunoblotting of Cytoskeletal Proteins
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Immunoblotting was performed as described previously [18] (link). Protein concentration was determined with the BCA protein assay kit (Pierce). Following antibodies were used: αSMA, α-tubulin, and ß-actin (Sigma-Aldrich) at 1∶5000 dilution; myosin light chain 2 (MLC2), phospho-MLC2 (Ser19), phospho-MLC2 (Thr18/Ser19), Src, phospho-Src (Tyr416), and vimentin (Cell Signaling Technology) at 1∶1000 dilution.
9
Antibody Panel for Signaling Pathway Analysis
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The following antibodies were used: PR65 (sc-15355, Santa Cruz, CA, USA), PP2Ac (05-421, Millipore), FLAG M2 (F1804, Sigma Aldrich), HA (sc-7392, Santa Cruz), phospho-AKT (Thr308) (9275, Cell Signaling), phospho-AKT (Ser473) (9271, Cell Signaling), phospho-SRC (Tyr416) (6943, Cell Signaling), phospho-FAK (Tyr397) (3283, Cell Signaling), phospho-PP2Ac (Tyr307) (ab32104, Abcam), SRC (2108, Cell Signaling), p21 (2946, Cell Signaling), β-actin (sc-4778, Santa Cruz), phospho-SAPK/JNK (Thr183, Tyr185) (9251, Cell Signaling), phospho-c-Jun (Ser73) (9164, Cell Signaling), phospho-c-Jun (Ser63) (9261, Cell Signaling), and c-Jun (sc-1694, Santa Cruz).
10
Antibody Validation for Protein Interaction Studies
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Antibodies against Csk (#2109 and #4980), phospho-Src (Tyr 416) (#2101; used for detecting human Src pY419), phospho-Src (Tyr 527) (#2105; used for detecting human Src pY530), His-Tag (#2366) were purchased from Cell Signaling Technology. Anti-Csk antibody (#17720–1-AP) for immunoprecipitation was purchased from proteintech. Antibodies against GAPDH (#ab37168), SUMO1(#ab32058) were from Abcam. Anti-Flag (M2), anti-HA and anti-Myc antibodies were from Sigma-Aldrich. Protein A/G PLUS Agarose beads (#K0115) were obtained from Santa Cruz Biotechnology. Ni2+-NTA agarose beads were from Qiagen. Glutathione Sepharose 4B beads (#17–0756-01) were from GE Healthcare Life Sciences. Puromycin (#P8833) and EDTA-free Protease Inhibitor Cocktail were from Sigma-Aldrich.
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