Mouse anti lamin a c

The cells were fixed with 4% paraformaldehyde for 15 min. After several washes, they were blocked with 0.01M phosphate-buffered saline supplemented with 1% BSA and 0.3% Triton X-100 for 30 min. Primary antibodies (mouse anti-LAP2α, 1:500, Abcam, UK; mouse anti-betaIII tubulin, 1:500, Abcam; mouse anti-NeuN, 1:1000, Abcam; rabbit anti-NeuN, 1:200, Abways; rabbit anti-Tbr1, 1:500, Abcam; mouse anti-γH2AX, 1:250, Millipore, USA; mouse anti-Lamin A/C, 1:200, Abcam; rabbit anti-H3K9me3, 1:4000, Abcam; mouse anti-6E10, 1:200, Abcam) were diluted with blocking buffer and applied overnight at 4° C. The cells were stained with suitable Alexa Fluor–labeled secondary antibodies (Invitrogen) in blocking buffer at a concentration of 1:500 for 1.5 h at room temperature. 4', 6-Diamidino-2-phenylindole (Thermo Fisher, IL, USA) was applied to counterstain cells for visualizing nuclei at 1:1000 in Milli Q water (Biocel, Millipore, USA). The images were obtained with an Olympus IX71 microscope using a Hamamatsu ORCA CCD camera and Leica TCS SP5.

For immunoblot analysis, cells were lysed in a cell lysis buffer containing 50 mM Tris, pH 8, 150 mM NaCl, 1 mM EDTA, 10% (vol/vol) glycerol, 1% (vol/vol) Triton X-100, and 0.05% (vol/vol) NP-40 supplemented with protease inhibitors (Roche). Proteins were then quantified using a bicinchoninic acid protein assay kit (Pierce). For immunoblotting of phosphorylated proteins, the cell lysis buffer was also supplemented with phosSTOP phosphatase inhibitor cocktail tablets (Roche), and samples were subjected to SDS-PAGE immediately after lysis. The following primary antibodies were from the indicated sources: mouse anti-α-tubulin, Upstate Biotech; mouse anti-pIκBα (S32/36), Cell Signaling Technology; mouse anti-total IκBα (tIκBα), a kind gift from Ron Hay, University of Dundee, Dundee, United Kingdom; mouse anti-p65, Santa Cruz; and mouse anti-lamin A/C, Abcam. Mouse anti-D8 monoclonal antibody AB1.1 (33 (link)) and the rabbit anti-A49 polyclonal antiserum (24 (link)) have been described previously. Primary antibodies were detected with goat anti-mouse/rabbit IRdye 800CW infrared dye secondary antibodies, and membranes were imaged using an Odyssey infrared imager (LI-COR Biosciences).