BEAS-2B ECs were lysed and protein concentrations were determined by the DC protein assay (modified Lowry assay from Bio-Rad Laboratories). Samples containing 40–50 μg EC lysate protein were separated by SDS-PAGE using 8% gels (for phosphorylated proteins), and those containing 10 μg AM lysate protein or 20–30 μg AM CM (or mouse lavage fluid after 100 kD filtration) protein were separated on 12.5% gels (for SOCS3 proteins) and then transferred to nitrocellulose membranes. After blocking with 4% BSA, the membranes were probed overnight with antibodies against phospho- and total STAT3 (both Cell Signaling, 1:1000), phospho-STAT6 (Cell Signaling, 1:500), β-actin (Sigma-Aldrich, 1:10,000) or SOCS3 (Abcam, 1:750). Films were developed using ECL detection (Amersham Biosciences) after incubation with peroxidase-conjugated secondary antibody (Cell Signaling). Relative band densities were determined by densitometric analysis using Image J software.
Phospho and total stat3
Manufactured by Cell Signaling Technology
Phospho- and total STAT3 is a laboratory reagent used to detect and quantify the levels of phosphorylated and total STAT3 protein in biological samples. STAT3 is a transcription factor that plays a crucial role in various cellular processes, including cell growth, differentiation, and immune response.
Automatically generated - may contain errors
Lab products found in correlation
Phospho stat6, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Epha2, by Cell Signaling Technology (1 mentions)
Erbb2, by Cell Signaling Technology (1 mentions)
Pdgfra, by Cell Signaling Technology (1 mentions)
Ptyr 4g10, by Merck Group (1 mentions)
Beta actin, by BD (1 mentions)
Smad4, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Western lightning plus ecl kit, by PerkinElmer (1 mentions)
2 protocols using phospho and total stat3
1
BEAS-2B EC Protein Quantification and Western Blotting
2
Western Blot Analysis of Cellular Signaling
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Cell pellets were lysed in the buffer described [19 (link)]. Proteins were separated on SDS-PAGE gels, transferred to PVDF membranes and immunoblotted with the following antibodies (Supplementary Table 3 ): phospho- and total STAT3, ERK, AKT, EPHA2 (Tyr588 and Ser897), ERBB2 (Tyr1221 and Tyr877), PDGFRA, SRC, p38, S6K1B, S6, BAD, cleaved caspase3 (all from Cell Signaling, Danvers, MA), pTyr 4G10 (Millipore, Billerica, MA), SHP-1 (kind gift from Dr. A. Veillette), mouse CC1 (kind gift from Dr. K.V. Holmes), human CEACAM1 (Millipore, Billerica, MA), CEA (kind gift from Dr. C. P. Stanners), CEACAM6 (BioLegend, San Diego, CA), SMAD4 (Santa Cruz, Dallas, TX) and beta-Actin (BD Biosciences). The secondary HRP anti-rabbit or -mouse antibodies (GE Healthcare, Baie-D’Urfe, Quebec) were detected using the Western Lightning Plus-ECL kit (Perkin-Elmer, Waltham, MA).
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