Mouse monoclonal anti lmna

Immunofluorescence stainings were performed using protocol reported previously (Vinaiphat and Thongboonkerd, 2018 (link), Yoodee et al., 2021 (link)). Briefly, the cells were fixed with 4 % paraformaldehyde/PBS at 25 °C for 15 min, permeabilized with 0.1 % Triton X-100/PBS at 25 °C for 15 min, and then washed with membrane preserving buffer (PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂). The cells were incubated with 5 % bovine serum albumin (BSA)/PBS at 25 °C for 30 min to block non-specific binders and then with mouse monoclonal anti-LMNA (Santa Cruz Biotechnology) or mouse monoclonal anti-ZO-1 (Invitrogen) antibody (both at 1:50 in 1 % BSA/PBS) at 37 °C for 1 h. After three washes with PBS, the cells were incubated with goat anti-mouse IgG conjugated with Alexa Fluor 488 (green) (Invitrogen) (1:5,000) mixed with Hoechst dye (Invitrogen) (1:1,000) in 1 % BSA/PBS at 37 °C for 1 h. After extensive washes with PBS and mounting on a glass slide, the cells were examined and imaged under Eclipse 80i fluorescence microscope (Nikon). Quantitative intensity data were measured from at least 100 cells in ≥ 10 random high-power fields (HPFs) per each biological sample using NIS-Elements D V.4.11 (Nikon). Using this software, distribution of the immunofluorescence signal was analyzed in its spectral format to localize protein expression.

Western blotting was performed as described previously (Fong-ngern et al., 2017a (link), Fong-ngern et al., 2017b (link)). Briefly, cellular proteins (30 μg/lane) were resolved by 12 % SDS-PAGE and transferred to a nitrocellulose membrane (EMD Millipore; Billerica, MA) using a semi-dry transfer unit (GE Healthcare; Uppsala, Sweden) at 85 mA for 1.5 h. After blocking non-specific binders with 5 % skim-milk/PBS for 1 h, the membrane was incubated overnight at 4 °C with mouse monoclonal anti-LMNA (Santa Cruz Biotechnology) (1:1,000), mouse monoclonal anti-ZO-1 (Invitrogen; Eugene, OR) (1:1,000), or mouse monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology) (1:2,000) antibody in 1 % skim milk/PBS. After three washes with PBS, the membrane was incubated with rabbit anti-mouse IgG conjugated with horseradish peroxidase (1:20,000 in 1 % skim-milk/PBS) (Sigma-Aldrich) at 25 °C for 1 h. Immunoreactive protein bands were visualized by SuperSignal West Pico chemiluminescence substrate (Pierce Biotechnology, Inc.; Rockford, IL) and quantified by using ImageQuant TL software (GE Healthcare).