Hpa010856

Primary human airway epithelia were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 (Thermo Scientific). Nonspecific binding was blocked by incubating the epithelia with Superblock blocking buffer (Thermo Scientific) for 1 h. To determine the localization of FXYD3, the epithelia were incubated with three primary antibodies: 1:1,000 mouse anti-ATP1A1 (Sigma-Aldrich; 05–369-25UG, Lot 3484974), 1:50 rabbit anti-FXYD3 (Sigma-Aldrich; HPA010856; Lot A57803), and 1:50 Phalloidin conjugated with Alexa-fluor 647 (Thermo Fischer) overnight at 4°C. Then, the primary antibodies were washed, and epithelia were incubated with conjugated secondary antibodies: goat anti-mouse-Alexa-488 and goat anti-rabbit-Alexa-568, both at 1:1,000 dilution, for 1 h at room temperature protected from light. The secondary antibodies were washed, and the epithelia were mounted onto glass slides with Vectashield mounting medium with DAPI for nuclear staining (Vector Laboratories).

Samples were separated by SDS-PAGE and transferred to PVDF membranes (Immobilon-FL, Millipore). The total protein for each lane was detected with revert total protein stain and washed as per manufacturer’s protocol (LI-COR). Following total protein detection, the membrane was blocked with 0.1% casein protein in PBS for 1 h at room temperature. Then, the membrane was concurrently probed with rabbit Na/K ATPase (1:1,000 dilution; Abcam; ab76020; Lot GR3184452‐8) and rabbit FXYD3 (1:250 dilution, Sigma-Aldrich; HPA010856; Lot A57803) antibodies for 2 h at room temperature. Following PBS washes, the membrane was incubated with a 1:10,000 dilution of anti-rabbit secondary antibody (LI-COR). After a final PBS wash, images were obtained with an Odyssey gel imager (LI-COR), and raw signal intensities were analyzed with ImageJ (v2.3.0/1.53f, NIH) using the approach of the NIH ImageJ user guide. We used the area under both bands observed with the Na/K ATPase for analysis because heating the sample induces Na/K ATPase dimers (11 (link)). Total protein staining was the gel loading control, and data are presented as the %FXYD3 siRNA normalized to its donor-matched siRNA control.