Snap-frozen LV tissue samples were homogenized and lysed in a buffer containing: 25 mM Tris–HCl, 150 mM NaCl, 2 mM EGTA, 5 mM EDTA, 0.5% NP-40 with protease and phosphatase inhibitor cocktails (Sigma-Aldrich, UK). For cell lysates, the buffer composition was: 25 mM Tris–HCl, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 0.1% sodium deoxycholate with protease and phosphatase inhibitor cocktails. Protein concentration was estimated using Bradford reagent (Sigma-Aldrich, UK). Tissue homogenates were separated by SDS/PAGE and transferred onto nitrocellulose membranes. Membrane fractions were obtained by centrifugation of heart lysates13 (link) and further processed as described earlier. The following primary antibodies were used: phospho-Akt (S473), total Akt, phospho-Erk1/2 (Thr202/Tyr204), total Erk1/2, phospho-ribosomal protein S6 (S235/236), total ribosomal protein S6, phospho-Src (Tyr416), total Src, total PP2Ac (all Cell Signaling); Nox2 (BD Transduction); Nox414 (link); eIF4E-BP1, caveolin-3 and phospho-PP2Ac (Tyr307) (Abcam); p47phox (EMD Millipore/Upstate); GAPDH and β-actin (Sigma). Imaging and densitometric quantification were undertaken either using enhanced chemiluminescence or with an Odyssey Li-Cor imaging system (Li-Cor Biosciences, UK).
Total src
Manufactured by Cell Signaling Technology
Sourced in United States
Total Src is a lab equipment product that measures the total amount of the Src protein in a sample. It provides a quantitative assessment of the total Src levels, regardless of its activation state or post-translational modifications.
Automatically generated - may contain errors
Lab products found in correlation
Total akt, by Cell Signaling Technology (5 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Phospho src, by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Total stat3, by Cell Signaling Technology (3 mentions)
Total erk1 2, by Cell Signaling Technology (2 mentions)
Phospho erk1 2, by Cell Signaling Technology (2 mentions)
Odyssey clx imager, by LI COR (2 mentions)
Src py416, by Cell Signaling Technology (2 mentions)
Py397 fak, by Abcam (2 mentions)
Total fak, by BD (2 mentions)
Total p130cas, by BD (2 mentions)
Py410p130cas, by Cell Signaling Technology (2 mentions)
Ps63c jun, by Cell Signaling Technology (2 mentions)
Py861fak, by Thermo Fisher Scientific (2 mentions)
Total c jun, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Cleaved parp, by Cell Signaling Technology (2 mentions)
Phospho p38, by Cell Signaling Technology (2 mentions)
Total p38, by Cell Signaling Technology (2 mentions)
16 protocols using total src
1
Protein Extraction and Western Blot Analysis
2
Western Blotting Analysis of Signaling Proteins
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Western blotting was performed as previously described (36 ) using the following antibodies: total FAK (BD Bioscience:610087), total p130Cas (BD Bioscience:610271); pY397FAK (Abcam, Cambridge, MA:ab81298); pY416Src (Cell Signaling, Danvers, MA:2101), total Src (Cell Signaling:2109), pY410p130Cas (Cell Signaling:4011), total c-Jun (Cell Signaling:2315), pS63c-Jun (Cell Signaling:2361); pY861FAK (Invitrogen, Carlsbad, CA:44–626G); α-tubulin (CALBIOCHEM, Burlington, MA:CP06). Phosphorylated protein expression was normalized to total protein for quantification using the Odyssey CLx imager (Li-Cor).
3
Western Blot Analysis of Signaling Pathways
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Protein lysates were prepared from cell lines or frozen tissues using RIPA buffer (Thermo Fisher Scientific, Waltham, MA) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific). Western Blot analyses were performed with standard techniques using the following antibodies: β-Actin, total Src, phosphoSrc (T416), phospho-ERK1/2 (T202/204), total and phospho-Akt (S473), total and phospho-S6 (S240/244), phospho-mTOR (S2448), phospho-4EBP1(T37/46), LC3B (all from Cell Signaling Technology), goat anti-human Osteoactivin/GPNMB antibody, p62 (both R&D Systems). Protein bands where visualized with IRDye secondary antibodies diluted in Odyssey blocking buffer containing 0.2% Tween and 0.1% SDS and signals were visualized with an Odyssey imager and analyzed with Image Studio software (all LI-COR Bioscience).
4
Comprehensive Western Blot Analysis
5
Western Blotting Protein Expression Analysis
6
Monoclonal Antibody-Based Protein Interaction Analysis
7
Western Blot Analysis of Signaling Proteins
8
Antibody Characterization for Stem Cell Research
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Polyclonal antibodies against CR1 used for immunofluorescence and flow cytometry analyses were produced by immunizing rabbits with purified recombinant soluble CR1 produced in 293 cells.40 (link) Monoclonal antibodies against Lgr5 (both conjugated and unconjugated) and CD133 (AC133 epitope) were obtained from Miltenyi Biotec (Bergisch Gladbach, Germany). Antibodies against CD44v6, Nanog, Musashi, Sox2 and EphB2 (the latter both Alexa Fluor 488-conjugated and unconjugated) were obtained from R&D System Inc. (Minneapolis, MN, USA). Anti-CK20 was from Dako (Carpinteria, CA, USA). Alexa Fluor-conjugated secondary antibodies were obtained from Invitrogen Molecular Probes. Anti-Alk4, cyclin D1, Glypican-1 and TIM23 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal anti-β-actin, GAPDH and PARP were from Sigma-Aldrich. Total Src, p-Src, p-AKT, total AKT, p-smad2, smad2, CD44, polo-like kinase 1, Bmi1 and β-catenin were from Cell Signaling Technology (Danvers, MA, USA). Secondary anti-mouse and anti-rabbit antibodies coupled to horseradish peroxidase were from GE Healthcare (Uppsala, Sweden).
9
Cellular Protein Profiling with Immunodetection
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Cell lysates were collected using Nonidet-P40 or radioimmunoprecipitation (RIPA) lysis buffer, supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Scientific), and immunodetection of electrophoresis-resolved proteins was performed using standard protocols. The E-Cadherin, vimentin, Snail, phospho-AKT, total AKT, phospho-p38, total p38, phospho-pERK, total ERK, phospho-STAT3, total STAT3, phospho-Src, total Src, phospho-pFAK, total pFAK, phospho-ACK-1, total pEPHB1, cleaved PARP, pTyr-1000, and GAPDH antibodies were from Cell Signaling Technology. Total ACK-1, phospho-EPHB1 antibodies were from Abcam, and the PARP antibody was from eBioscience. Kinomeview immunoblotting was performed using the pTyr-1000 antibody from Cell Signaling Technology.
10
Western Blot Analysis of Signaling Proteins
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Western blotting was performed as described previously (Herrero-Gonzalez et al., 2010 (link)). In brief, equivalent amounts of proteins (20 μg per lane) were separated on NuPAGE Novex Bis-Tris (4%–12%) midi gels (Life Technologies). The proteins were transblotted using an iBlot dry blotting system (Life Technologies). After blocking, the membranes were incubated overnight at 4°C with the primary antibodies against Y416 Src (1:200; Cell Signaling, Danvers, MA, USA; Ref. 2101), total Src (1:500; Cell Signaling; Ref. 2108), PTEN (1:500; Cell Signaling; Ref. 9556S), Y397 FAK (1:1,000; Life Technologies; Ref. 44-624G), Y576 FAK (1:500; Life Technologies; Ref. 44652G), Y577 FAK (1:500; Life Technologies; Ref. 44-614G), and total FAK (1:500; Life Technologies; Ref. AHO0502). The antibodies against glyceraldehyde phosphate dehydrogenase (GAPDH, 1:15,000; Ambion, Thermo Fisher Scientific; Ref. AM4300) or alpha-actinin (1:1,000; Chemicon International, Merck Millipore; Ref. MAB1682) were used as a loading control. After extensive washing, the membranes were incubated with peroxidase-conjugated anti-rabbit IgG or the anti-mouse IgG antibody (Santa Cruz Biotechnology, Dallas, TX, USA; Refs. sc-2030 and sc-2005) in TTBS and developed with a chemiluminescent substrate (Western Blotting Luminol Reagent; Santa Cruz Biotechnology). X-ray films were obtained from Fujifilm (Madrid, Spain).
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