Where required, cell lysates were prepared as previously established (21 (link)). For supernatant samples, 4 × SDS loading buffer was added and samples were analyzed following the same protocol. Cell-lysate western blot samples were probed for human TN-C (N-Terminal, mab1908, Merck Millipore, California, USA), Histidine-Tag (H1029, Sigma-Aldrich), and β-actin (A5316, Sigma-Aldrich), with TN-C expression normalized to β-actin, whilst supernatant samples were analyzed for TN-C only. Mouse BALF was concentrated by trichloroacetic acid (TCA, Sigma-Aldrich) precipitation prior to analysis and TN-C expression determined using mouse TN-C antibody (N-Terminal, T3413, Sigma-Aldrich). sEV isolates were analyzed for sEV-enriched proteins cluster of differentiation 9 (CD9, sc-13118, Santa-Cruz Biotechnology, Dallas, USA) and flotillin-1 (ab13493, Abcam, Cambridge, UK), with the negative control glucose regulated protein 94 (GRP94, ab7291, Abcam) utilized to confirm the lack of cellular protein contamination. Densitometrical analysis was performed using ImageJ software (Version 1.5i; NIH). Due to multiple variants of TN-C being expressed, the dominantly expressed band was measured for each experiment.
Ab13493
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab13493 is a laboratory antibody product available from Abcam. It is a primary antibody used in various research applications. The core function of this product is to specifically bind to and detect the target antigen.
Automatically generated - may contain errors
2 protocols using ab13493
1
Western Blot Analysis of Tenascin-C
2
Quantification of Flotillin-1 and TSG101 Proteins
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Protein concentrations were determined by Bradford dye assays. Ten microgram proteins were resolved on a 12% polyacrylamide gel, separated by polyacrylamide gel electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). Western blot analysis was performed using goat polyclonal antibody anti-Flotillin 1 (Flot-1) (1:500 dilution; ab13493 Abcam, Cambridge, UK) and rabbit monoclonal antibody anti-Tumor Susceptibility 101 (TSG101) (1:500 dilution; ab125011 Abcam, Cambridge, UK) as primary antibodies and anti-goat HRP conjugated IgG, and anti-rabbit HRP conjugated IgG as secondary antibodies at 1:1000 dilutions.
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