Snap-frozen paws or macrophages were processed in 1% NP-40 lysis buffer containing proteinase inhibitor cocktail (Sigma-Aldrich) and 50 mM sodium fluoride (Sigma-Aldrich) and protein lysates cleared by centrifugation. Equivalent amounts of protein were resolved on a 10% SDS-PAGE gel and transferred to PVDF membrane (Millipore). The membrane was probed using the following primary antibodies: rabbit anti-mouse LC3 (1:2,000, Cat # L7543, Sigma-Aldrich), phospho-AMPKα (1:1,000, Cat # 2535, Cell Signaling Technology), phospho-mTOR (1:1,000, Cat # 5536, Cell Signaling Technology), IKK-α (1:1,000, Cat # 2682, Cell Signaling Technology), IκB-β (1:200, Cat # SC-945, Santa Cruz), total p65 (1:2,000, Cat # 8242, Cell Signaling Technology), phospho-p65 (1:2,000, Cat # 3033, Cell Signaling Technology) and mouse anti-mouse p62 (1:2,000, Cat # MABC32, clone 11C9.2, Millipore) followed by the appropriate HRP-conjugated secondary antibodies. Goat anti-mouse actin (1:3,000, Cat # SC-1615, Santa Cruz) served as control for quantity and quality of protein.
Goat anti mouse actin
Manufactured by Santa Cruz Biotechnology
Goat anti-mouse actin is a primary antibody that specifically binds to actin, a key structural protein found in eukaryotic cells. This antibody is produced in goats and is reactive against the actin of mouse origin.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Sodium fluoride, by Merck Group (1 mentions)
Phospho p65, by Cell Signaling Technology (1 mentions)
Total p65, by Cell Signaling Technology (1 mentions)
Proteinase inhibitor cocktail, by Merck Group (1 mentions)
Phospho ampkα, by Cell Signaling Technology (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
2 protocols using goat anti mouse actin
1
Western Blot Analysis of Cell Signaling
2
Western Blot Protein Detection
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Cells were lysed in laemmli buffer containing 10% β-mercaptoethanol and boiled for 15mins at 95°C. The samples were then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Thereafter the membranes were blocked in 10% milk powder in TBST. Membranes were then incubated in the primary and secondary antibodies at RT for 1h each respectively, with washing in TBST in between. After washing the Supersignal West Pico Chemiluminescent substrate was added and the membranes were imaged on Chemidoc MP imaging system (Biorad). The following antibodies were used: goat anti-mouse p110γ (Santa Cruz Biotechnology), bovine anti-goat HRP (Santa Cruz Biotechnology), goat anti-mouse actin (Santa Cruz Biotechnology).
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