Mouse anti ha 11 epitope tag antibody

To immunolabel CFTR at the PM, CF HBE cells were transduced with wt-CFTR or F508del-CFTR containing a 3HA-tag in the fourth extracellular loop of CFTR. Confluent cells were gently washed with PBS and incubated with the mouse anti-HA.11 epitope tag antibody (1:400, Biolegend, cat. #901514) at 4°C for 45 min, then rinsed with PBS and fixed in 4% methanol-free formaldehyde (Thermo Fisher Scientific) for 15 min at room temperature. The fixed cells were exposed to goat anti-mouse-IgG conjugated to Alexa Fluor 488 secondary antibody (Invitrogen; 1:1000 dilution) for 1 h, washed with PBS and mounted in ProLong Diamond Antifade Mountant (Invitrogen) for imaging. The antibody was validated in cells that do not express any 3HA-tagged protein. For FLNA immunolabeling, and following FLNA knockdown, HBE cells transduced with wt-CFTR were gently washed with PBS, fixed, permeabilized (0.5% Triton X-100), blocked for 1 h (2% BSA) and incubated with the rabbit anti-FLNA antibody (1:100, Invitrogen, cat. #PA5-86143) for 2 h. Cells were then exposed to goat anti-rabbit IgG conjugated to Alexa Fluor 594 secondary antibody (Invitrogen; 1:1000 dilution) for 1 h and washed and mounted in PBS for imaging. The low fluorescence detected in cells due to the knockdown of FLNA (compared to the scrambled control) validated the antibody.