Anti myc monoclonal antibody 9e10

In vitro chromatin-released fraction prepared from the isolated chromatin of TRF2ts MEFs was diluted with IP buffer (20 mm Tris-HCl, pH 7.4, 0.5% Nonidet P-40, 150 mm KCl, 1 mm MgCl₂, 0.5 mm EDTA, and protease inhibitor cocktail) and incubated with 1 μg of anti-Myc monoclonal antibody (9E10, Santa Cruz Biotechnology) for 2 h at 4 °C. Immunoprecipitates were prepared by incubation with Dynabeads protein G (Invitrogen) and subjected to immunoblotting with the indicated antibodies. Cell extracts prepared from NIH-3T3 cells that were transfected with Basic-NLS-EGFP, Basic-RA-NLS-EGFP, and NLS-EGFP expression plasmids using radioimmunoprecipitation assay buffer without SDS (Nacalai Tesque) were incubated with 1 μl of anti-GFP polyclonal antibody (Abcam) for 2 h at 4 °C. Immunoprecipitates were prepared by incubation with Dynabeads protein A (Invitrogen) and subjected to silver staining and immunoblotting with the indicated antibodies. Silver staining was performed using the Sil-Best Stain One kit (Nacalai Tesque), according to the manufacturer's instructions.

Preparation of protein extracts and Western blot analysis were performed as described previously^{9 (link)}. Anti-phospho-p38 MAPK monoclonal antibody D3F9 (Cell Signaling), anti-Hog1 polyclonal antibody y-215 (Santa Cruz), anti-phospho-p44/42 MAPK polyclonal antibody (Cell Signaling), anti-Mpk1 polyclonal antibody yN-19 (Santa Cruz), anti-Myc monoclonal antibody 9E10 (Santa Cruz) and anti-Mcm2 polyclonal antibody N-19 (Santa Cruz) were used. Detection was carried out by using a LAS-4000 (Fuji Film) with Immobilon Western (Merck Millipore) or the Odyssey Imaging Systems (LI-COR Biosciences). Signal intensities were quantified by the Odyssey Imaging Systems, and statistical analysis was performed with Excel (Microsoft).

Polyclonal anti-ROCK I (H-85) and anti-ROCK II (H-85) antibodies and monoclonal anti-Myc (9E10) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-α-actinin antibody and monoclonal anti-MLC pS19 antibody were purchased from Cell Signaling Technology (Danvers, MA). Monoclonal anti-MLC (MY-21) antibody, monoclonal anti-MLCK (K-36) antibody, gelatin, BDM, and ML-7 were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-facsin antibody, Y27632, puromycin, and fibronectin were purchased from EMD Millipore (Billerica, MA). Alexa Fluor 488-phalloidin, the Alexa Fluor 546 protein labeling kit, Alexa Fluor 488- and Alexa Fluor 546-conjugated secondary antibodies, and Lipofectamine were purchased from Thermo Fisher Scientific (Waltham, MA). The horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The plasmid pCS2+ -GFP-UtrCH was purchased from Addgene (Cambridge, MA). The plasmid pEGFP-actin was purchased from Takara Bio USA, Inc. (Mountain View, CA). The plasmid pEGFP-N1-MLCK was described previously^{28 (link)}. The plasmids mCherry-UtrCH, pEF-Myc-ROCK II C.A. (a.a 6–553), and pEF-Myc-ROCK II K121G (a.a 6–553) were kindly provided by Dr. H. H. Lee (National Yang-Ming University, Taiwan).