Flash-frozen skeletal muscle was homogenized in a lysis buffer (Scalzo et al. 2018b (link)) then centrifuged (18,000 g for 10 min at 4°C). The supernatant was analysed for protein concentration by Bradford assay. Citrate synthase enzyme activity was determined by previously described methods (Keller et al. 2015 (link)). Protein in Laemmli sample buffer was run on 4%–20% Tris-HCl gels. Proteins were electrophoretically transferred to PVDF membranes, and equivalence of protein loading was assessed by staining of membrane-bound proteins by Ponceau S stain and the protein vinculin. Blots were probed using antibodies against mitochondrial OXPhos complexes (Abcam #ab110412), CD31 (Cell Signalling #3528S), vascular endothelial growth factor (VEGF; Abcam #ab46154), and vinculin (Cell Signalling # 4650s); all antibodies were used at a concentration of 1:1000 overnight at 4°C and followed by fluorescent secondary. Proteins were detected by Li-COR (Odyssey CLX) Western blot scanner, and densitometric analysis was performed using Image Studio v4.1. In addition to the above-mentioned visual confirmation of equal protein loading and transfer across membranes with ponceau staining and the protein-loading control vinculin, a sample-loading control (human skeletal muscle) was used across all blots and all data were normalized to that lane.
Ab46154
Manufactured by Cell Signaling Technology
Ab46154 is a primary antibody produced by Cell Signaling Technology. It is a recombinant rabbit monoclonal antibody that recognizes a specific protein target. The core function of this product is to bind and detect the target protein in biological samples for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Beyotime (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Cell lysis buffer, by Beyotime (1 mentions)
Gapdh, by Bioworld Technology (1 mentions)
Ab31334, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Wbkls0500, by Bio-Techne (1 mentions)
Flour chem fc2 imaging system, by Bio-Techne (1 mentions)
2 protocols using ab46154
1
Skeletal Muscle Protein Analysis
2
Western Blot Analysis of Immune Signaling Proteins
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Cells were homogenized in cell lysis buffer (Beyotime), and protein concentration was determined using a BCA kit (Beyotime). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with specific primary antibodies. Then the membrane was incubated with appropriate HRP-conjugated secondary antibodies. Signals were monitored by an enhanced chemiluminescence reagent (Millipore, WBKLS0500) and subjected to Alpha Innotech Flour Chem-FC2 imaging system (Alpha Innotech). The antibodies used in this study are as follows: VEGF (abcam, ab46154), RIG-I (Cell signaling technology, 3743), MAVS (abcam, ab31334), NOXA (abcam, ab13654), GAPDH (Bioworld Technology, MB001), β-actin (Cell signaling technology, 4967).
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