Anti ha antibody 16b12

The subcellular localization of the HBH-CFTR-3HA variant in CFBE Tet-on cells was examined as reported previously [74 (link)]. The cells, plated on a coverslip, were washed twice with a phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (FUJIFILM-Wako Chemicals, Japan) for 30 min at room temperature (RT). Cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min and blocked with 0.5% bovine serum albumin (BSA) in PBS for 20–30 min at RT. Cells were incubated for 2 h at RT with an anti-HA antibody (16B12, BioLegend, San Diego, CA, USA, 1:1000) in 0.5% BSA in PBS. Then, the cells were washed three times with PBS, and an Alexa Fluor^® 488–conjugated anti-mouse IgG (1:500, Jackson ImmunoResearch, West Grove, PA, USA) was added to the cells, which were then incubated for 1 hour at RT. After washing the cells three times with PBS, a DAPI solution (Peptide Institute, Inc., Japan) diluted in PBS (1:5000) was added to the cells, which were then incubated for 5 min at RT. After washing the cells three times with PBS, the cells were mounted with a Vectashield^® mounting medium (Vector Laboratories, Newark, CA, USA). The images were visualized and captured with an inverted laser confocal fluorescence microscope (SP8, Leica Microsystems GmbH, Germany).

HEK293 cells were grown on coverslips coated with poly-l-lysine and transfected as described above. One day after transfection, cells were washed once with PBS, blocked in 200 μL PBS containing 2% BSA before incubation overnight at 4 °C with anti-HA antibody (16B12, BioLegend) diluted 1:500 in PBS, 1% BSA. The next morning, cells were washed twice with PBS for 15 min followed by 1 h at room temperature incubation with donkey anti-mouse secondary antibody conjugated to Alexa Fluor 549 (ThermoFisher Scientific) diluted 1:500 in PBS, 1% BSA followed by two additions 15 min washes in PBS. Cells were mounted onto coverslips using Prolong Gold antifade reagent containing 4′,6-diamidino-2-phenylindole (DAPI; ThermoFisher Scientific). Confocal imaging was conducted using a Leica TCS SP5 II Basic VIS system (Leica Microsystems).

The PM density and stability of the HBH-CFTR-3HA variants in CFBE Tet-on cells on 24-well plates were measured by cell surface ELISA as reported previously [43 (link)]. Briefly, cell surface CFTR-3HA was labeled with anti-HA antibody (16B12, BioLegend, San Diego, CA, USA), biotinylated anti-mouse IgG (Funakoshi, Japan), and NeutrAvidin protein-HRP (Thermo Fisher, Waltham, MA, USA) and detected by the Amplex Red reagent (Thermo Fisher, Waltham, MA, USA). For the PM stability measurement, Cd or PYO was added to the cell culture medium during the 4 h’ chase at 37 °C incubation.