Embryos were stained as described previously [21 (link)]. The following primary antibodies were used: mouse monoclonal anti E-cadherin (BD Biosciences, 1:250), anti β-catenin (BD Biosciences, 1:250), anti-phosphotyrosine (Cell Signaling, 1:500), anti-PrP 6H4 (Prionics, Switzerland); rabbit polyclonal anti β-catenin (Sigma, 1:500) and anti-Src (Cell Signaling, diluted 1:100). Secondary antibodies: Alexa-488 conjugated goat anti-rabbit or -mouse and Cy3-conjugated donkey anti-rabbit or -mouse (Jackson Immunoresearch, 1:1000), Cy5 conjugated goat anti-rabbit or -mouse (Invitrogen, 1:1000). Whole embryos were imaged as flat (deyolked) or thick (with yolk) mounts on LSM 510 and 710 confocal microscopes (Zeiss), and images were further processed using Adobe Photoshop CS5. Differences in protein distribution along embryonic axes were visualized using fluorescence profiles generated with LSM 510 and ZEN software (Zeiss). To study β-catenin translocation, Z-sections of whole embryos were generated and marginal cells with nuclear β-catenin were counted. To determine ratios of plasma membrane vs. cytosolic β-catenin in dorsal blastomeres, whole cell/cytoplasm areas were outlined and the corresponding fluorescence (integrated densities) measured and subtracted using Image J.
Anti phospho tyrosine
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-tyrosine is a laboratory reagent that detects phosphorylation of tyrosine residues in proteins. It is used to study signal transduction and protein regulation in cells.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 510, by Zeiss (2 mentions)
Anti β catenin, by Merck Group (1 mentions)
Anti src, by Cell Signaling Technology (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti e cadherin, by BD (1 mentions)
Epha2, by Cell Signaling Technology (1 mentions)
P tyr 1000, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti phospho egfr tyr1068, by Cell Signaling Technology (1 mentions)
Anti beta actin antibody sc 1615, by Santa Cruz Biotechnology (1 mentions)
Anti phospho, by Cell Signaling Technology (1 mentions)
Anti phospho met tyr1234 1235, by Cell Signaling Technology (1 mentions)
C jun, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Genistein, by Merck Group (1 mentions)
U73122, by Merck Group (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti caldesmon, by Merck Group (1 mentions)
Anti mypt, by Cell Signaling Technology (1 mentions)
15 protocols using anti phospho tyrosine
1
Embryo Immunofluorescence Staining and Analysis
2
Antibody Panel for Cellular Signaling Analysis
3
Mechanisms of Local Anesthetic Action
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All drugs used in this experiment were the highest commercially available purity. Bupivacaine and lidocaine were purchased from Reyon Pharmaceutical Co., Ltd. (Seoul, Korea). Mepivacaine was obtained from Hana Pharmaceutical Co., Ltd. (Gyeonggi-do, Korea). Sodium orthovanadate, genistein, U-73122, SP600125, PD98059 and anti-β-actin antibody were obtained from Sigma-Aldrich (St. Louis, MO, USA). Y-27632 was obtained from Calbiochem (La Jolla, CA, USA). Anti-phospho-tyrosine, anti-phospho-PLC γ-1 (Tyr783), anti-phospho-JNK (Thr183/Tyr185), anti-phospho-ERK (Thr202/Tyr204), anti-phospho-MYPT (Thr696), anti-PLC γ-1, anti-JNK, anti-ERK and anti-MYPT antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-phospho-caldesmon (Ser789) and anti-caldesmon antibodies were obtained from Millipore (Billerica, MA, USA) and Abcam (Cambridge Science Park, Cambridge, England), respectively. Fura-2/AM was purchased from Molecular Probes (Eugene, OR, USA). All concentrations are expressed as the final molar concentration. genistein, U-73122, SP600125 and PD98059 were dissolved in DMSO. Unless otherwise stated, all drugs were dissolved and diluted in distilled water.
4
RET Kinase Domain Autophosphorylation Kinetics
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The time course of autophosphorylation of recombinant purified RET KD (2.5 μM) was examined in the presence of saturating concentrations of ATP (5 mM) and MgCl2 (10 mM) for 0–80 min as previously described18 (link). Reactions were stopped by addition of 4× loading sample buffer (Invitrogen) with 10% β-mercaptoethanol and boiling for 5 min. Samples were then loaded onto to a NuPAGE Invitrogen 4–12% Bis-Tris precast gel. Phosphorylation was detected with the following antibodies: anti-phospho-Tyr1062 RET (used at 3000-fold dilution), anti-phospho- tyrosine (used at 1000-fold dilution), phospho-Tyr 905 RET (used at 1000-fold dilution), and anti-total RET (used at 1000-fold dilution), purchased from Cell Signaling Technology. Uncropped gel data are supplied in Supplementary Fig. 9 .
5
Western Blot Analysis of Immunoprecipitated Proteins
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The same amounts of the immunoprecipitated proteins were isolated on 10% SDS polyacrylamide gels, Separation of the same amount of immunoprecipitation proteins by 10% SDS polyacrylamide gels was done, followed by transfer to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA) on a wet transfer apparatus (Bio-Rad) for 100 min at 90 V. The blocked membrane, treated with 5% nonfat milk, was subsequently incubated with the following specific primary antibodies: anti-phospho-tyrosine (#9411; Cell Signaling Technology), anti-PSF, or anti-Hakai (ab91185; Abcam) antibody with GAPDH as a reference at 4 °C overnight. After washing the membranes with TBST, the second antibody coupled with horseradish peroxidase (HRP) was incubated at room temperature for 2 h. Then, band detection, scanning, and band intensity analysis were detected using chemiluminescence kit (GE Healthcare, Freiburg, USA) and MultiSpectral Imaging System (EC3 410, UVP, Upland, CA, USA).
6
Protein Extraction and Immunoprecipitation
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Total proteins were extracted in cold lysis buffer containing 50 mM Tris, pH 7.5, 2 mM EDTA, 100 mM NaCl, 50 mM NaF, 1% Triton X-100, 1 mM Na3VO4 and 40 mM β-glycerol phosphate, with added protease inhibitor cocktail (Roche, Mannheim, Germany). For immunoprecipitation, equal amount of protein samples were precleared and incubated with 2 μg of antibody and 20 μl of 50% protein A/G agarose bead slurry (Pierce Biotechnology, Rockford, IL, USA) at 4 °C for overnight with gentle agitation. The beads were washed and boiled in 3× Laemmli buffer. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were developed with HRP-conjugated secondary antibodies and ECL Prime reagents (GE, Piscataway, NJ, USA). Signals were detected with a LAS-4000 luminescent image analyzer (Fujifilm, Stamford, CT, USA). The densitometry analysis was performed using Image-J software (NIH). The following antibodies were used: anti-phospho-tyrosine (#9411), Src (#2110), FAK (#3285), Vav2 (#2848), and p130Cas (#13383) were from Cell Signalling; anti-Src (ab109381) and Crk (ab133581) were from Abcam (Cambridge, UK); anti-paxillin (#05–417) was from EMD Millipore; anti-Tiam1 (AF5038) was from R&D Systems (Minneapolis, MN, USA); anti-DOCK180 (sc-6167) was from Santa Cruz (Dallas, Texas USA).
7
Western Blot Immunodetection Protocols
8
Western Blot Analysis of EGFR Signaling
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25 μg (A431 and Cos1) or 40 μg (lung cancer tissues) of lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Far-Western and Western blotting analyses were carried out as previously described [6 (link), 7 (link), 23 (link)]. Replica membranes were incubated with 200 nM GST fusion proteins in 5 % fat-free milk dissolved in TBST (150 mM NaCl, 10 mM Tris–HCl [pH 8.0], and 0.05 % Tween-20) for 2 h, washed for 20 min, and specific bands were visualized by chemiluminescence. Blots were stripped and reprobed with anti-EGFR (Santa Cruz Biotechnology), anti-phosphotyrosine (Cell Signaling), and anti-actin (Santa Cruz Biotechnology) antibodies. For quantitative far-Western analysis (Fig. 4 b), band intensities were quantitated using ImageJ densitometry software (National Institutes of Health). For estimation of the absolute amount of tyrosine phosphorylation (Fig. 3 and Additional file 1 : Figure S4), anti-phosphotyrosine Western blots were quantified using the LI-COR Odyssey IR detection system (LI-COR Biosciences).
9
Immunoblot Analysis of HER2 Signaling
10
Immunoblotting Techniques for Protein Analysis
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The following antibodies were used: anti-TG2 (Thermo Scientific, CUB7402), anti-PKM2 (Cell Signalling, D78A4), anti-EEF1A1 (Milipore, 05-235), anti-Hsc70 (Abcam, 51052), HSPA1A (Santa Cruz, 7947), anti-Biotin (Abcam, 1227), anti-p62/SQSTM1 (mbl, PM045), anti-LC3 (Novus Biologicals, NB100-2331), anti-GAPDH (Sigma, G9545), anti-Actin (Sigma, 2066), anti-Beclin1 (Santa Cruz, 10086), anti-HIF1-beta (Novus Biologicals, NB100-124), anti-ULK1(Santa Cruz, 33182), anti-phospho-tyrosine (Cell Signaling, 9411) and HRP-conjugated secondary antibodies (Bio-Rad Laboratories).
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