Immunofluorescence staining of MDCK cells

Cell culture medium was removed and cells were washed twice with 1X PBS. Then the cells were fixed in 4% Paraformaldehyde for 20 min. This was followed by washing twice with 1X PBS and then incubated in 0.5% Triton X-100 for 20 min. Then the cells were blocked for 20 min in 1% BSA (made in 1X PBS). The cells were then incubated overnight with the primary antibodies diluted in 1% BSA made in 1X PBS. Then primary antibody was removed and the cells were washed twice with 1X PBS followed by incubating in 1% BSA for 20 min. Then cells were incubated for 2–3 h in the secondary antibody. The following primary antibodies were used—mouse monoclonal antibody against Lamin A (Thermo Fisher,Cat#MA1-06101, 1:100 dilution), mouse monoclonal antibody against Lamin B1 (Abcam, #ab8982, 1:100 dilution), rabbit polyclonal antibody against ZO-1 (ThermoFisher, Cat#40-2200, 1:500 dilution), monoclonal antibody against LamA phosphorylated at Ser22 (Cell signaling, Cat#2026S 1:125 dilution). The following secondary antibodies were used at 1:500 dilution in 1% BSA: goat anti-mouse Alexa 647Fluo (ThermoFisher, Cat No. A28181) and goat anti-rabbit Alexa 488Fluo (ThermoFisher, Cat No. A-27034).