MEG-01 cells treated with 10 nM PMA alone or 10 nM PMA with 1 μM or 5 μM ANA for 2 days were peeled off, and suspended cells were collected after incubation for 1 hour at 37°C with 5% CO2. The suspended cells were lysed with SDS lysis buffer containing 1.7% SDS, 60 mM Tris-HCl, pH 6.8, 0.85% 2-mercaptoethanol, and proteinase inhibitor cocktail to examine the expression of FAK, tyrosine-phosphorylated FAK (pFAK), and phosphorylated MLC2 (pMLC2) by Western blotting. FAK, pFAK, and pMLC2 were detected with an anti-FAK antibody (EP695Y, Abcam, Cambridge, UK), anti-phospho-FAK (Tyr397) antibody (3283, Cell Signaling, Danvers, MA), and anti-phospho-MLC2 antibody (3675S, Cell Signaling, Danvers, MA), respectively. Protein loading of each well was assessed by an anti-β-actin antibody. The phosphorylation state of FAK was quantified as the density ratio of protein bands pFAK/FAK, and the phosphorylation state of MLC2 was quantified as the density ratio of protein bands pMLC2/β-actin.
Anti phospho fak tyr397 antibody
Manufactured by Cell Signaling Technology
Sourced in United Kingdom
The Anti-phospho-FAK (Tyr397) antibody is a research tool used to detect the phosphorylation of Focal Adhesion Kinase (FAK) at the Tyr397 residue. The antibody is designed to specifically recognize this post-translational modification, which is a key regulatory event in FAK signaling pathways.
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Lab products found in correlation
2 protocols using anti phospho fak tyr397 antibody
1
PMA and ANA Modulate FAK and MLC2 Phosphorylation
2
Western Blot Analysis of Phospho-FAK
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Whole-cell protein extracts were obtained by direct lysis in Laemmli buffer heated to 95 °C. Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes [47] . The membranes were incubated overnight at 4 °C with anti-phospho-FAK (Tyr397) antibody (#8556), anti-total FAK antibody (A-17;#13309) or anti GAPDH antibody (1:1000; #2118, Cell Signaling, Leiden, The Netherlands), and then with secondary anti-rabbit HRP-conjugated antibody (1:2000; Cell Signaling) for 1 hour at room temperature. Following incubation with HRP-conjugated secondary antibodies the chemiluminescence was visualized using the Novex ECL + kit (Novex® ECL, Invitrogen). Densitometry was performed on scanned immunoblot images using the ImageJ gel analysis tool.
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