Anti phospho mlc2 antibody

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.

H3K9/14ac and H3 antibodies were purchased from Upstate Biotechnology. Anti-UBF and anti-HA antibodies were from Santa Cruz. Anti-phospho-MLC2 antibody was obtained from Cell Signaling Technology. Anti-MLC, anti-BrU, anti-β-actin antibodies, doxycycline, and trichostatin A (TSA) were obtained from Sigma-Aldrich. Anti-myc antibody was purified from hybridoma clone 9E10. HDAC1 antibody was from Abcam. Y27632 and Blebbistatin were from Merck Biosciences.