Mouse monoclonal anti arid1a

Proteins were extracted from individual samples using Laemmli's buffer and their concentrations were measured by Bradford's method using Bio-Rad Protein Assay (Bio-Rad). Proteins with an equal amount (50 µg/sample/lane) were resolved by 12% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking non-specific bindings with 5% skim-milk/PBS for 1 h, the membrane was incubated at 4 °C overnight with mouse monoclonal anti-ARID1A (Santa Cruz Biotechnology) or mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology) (all were diluted 1:1,000 in 1% skim milk/PBS). After probing with corresponding secondary antibody conjugated with horseradish peroxidase (HRP) (Dako; Glostrup, Denmark) (diluted 1:2,000 in 1% skim milk/PBS) at 25 °C for 1 h, the immunoreactive protein bands were visualized by SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Inc.; Rockford, IL) and autoradiography. Band intensity data was obtained using ImageQuant TL software (GE Healthcare).

The cells were grown on a coverslip and treated as described above. After rinsing with PBS, the cells were fixed with 4% (v/v) paraformaldehyde/PBS at 25 °C for 15 min and then permeabilized with 0.1% Triton X-100/PBS at 25°C for 15 min. After washing, non-specific bindings were blocked with 1% BSA in PBS at 25 °C for 30 min and the cells were incubated at 4 °C overnight with mouse monoclonal anti-ARID1A (Santa Cruz Biotechnology) (1:50 in 1% BSA/PBS). After another washing step, the cells were incubated with corresponding secondary antibody conjugated with Alexa Fluor 488 (Invitrogen) (1:2,000 in 1% BSA/PBS) at 25 °C for 1 h. Nuclei were counterstained by Hoechst dye (Sigma-Aldrich) (1:2,000 in 1% BSA/PBS). Finally, the cells were extensively washed with PBS and mounted onto a glass slide using 50% glycerol in PBS. Cellular imaging was done by using Nikon Eclipse 80i fluorescence microscope (Nikon; Tokyo, Japan) and expression level of ARID1A was analyzed by measuring mean fluorescence intensity from at least 100 cells in ≥10 random high-power fields (HPFs) of each sample using NIS-Elements D V.4.11 (Nikon).