Alexa fluor 488 conjugated antimouse or rabbit igg

Cells were grown on a 96 well plate. By using BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD biosciences), cells were fixed in fixation buffer for 30 min and were permeabilized with permeabilization/wash buffer. After washing by PBS, the cells were incubated with the primary antibodies (anti‐YAP, anti‐OTUB1, and anti‐FLAG antibodies). As a secondary antibody, Alexa Fluor 488‐conjugated antimouse or rabbit IgG and Alexa Fluor 594‐conjugated antimouse or rabbit IgG were used (Invitrogen). DNA was visualized by hoechst33342 staining. Immunostaining of the cell preparations was recorded by using an epifluorescence Zeiss Axioplan 2 microscope (Zeiss, Inc.) attached to a charge‐coupled‐device camera.

Cells were suspended in ice-cold Lysis buffer (50 mM Tris-HCl, pH 7.5 with 150 mM NaCl, 5 mM EDTA, 1% NP-40, and 0.5% sodium deoxycholate) containing a protease inhibitor cocktail (Nacalai Tesque). Cell lysates were resolved by SDS-PAGE and analyzed by Western blotting as described previously [6] (link). The antibodies used were an anti-FLASH monoclonal antibody (1∶1000; generated in our laboratory [6] (link)), anti-FLASH polyclonal antibody (1∶1000; M-300; Santa Cruz), anti-actin (1∶5000; C4; Chemicon), anti-histone H3 (1∶1000; BioLegend), and anti-estrogen receptor (1∶100; HC-20; Santa Cruz). The secondary antibodies used were Alexa Fluor 488-conjugated anti-mouse or rabbit IgG (1∶5000; Invitrogen).